Compositions and methods for enhancing cognitive function

ABSTRACT

The invention encompasses novel compositions containing a methylation modifying compound isolated from botanical raw materials, including a fruit of the genus  Coffea , and microbial raw materials, methods of making such compositions, and methods of treating or preventing human disease using such compositions. The methylation modifying compound in botanical extracts of the present invention can dramatically increase the level of methylation of PP2A, thus increasing cognitive function, particularly in persons suffering from or prone to developing Alzheimer&#39;s disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 60/649,902 entitled “Enhancing Cognitive Function,”filed Feb. 3, 2005. The entire disclosure of this application isincorporated by reference.

GOVERNMENT RIGHTS

This work is supported at least in part by grants to Dr. Jeffry Stock.The government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to treatment or prevention of humandisease and to the enhancement of the general health or well-being of ahuman subject using compositions extracted from herbs and otherbotanical and microbial materials.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is one of the most prevalent of progressivebrain disorders. Currently an estimated 4.5 million older Americanssuffer from AD. According to the latest estimates of the current andfuture prevalence of AD, the number of older people with AD will growdramatically as the population ages. Projections indicate that as manyas 13 million older Americans will have AD by 2050 unless new ways arefound to prevent or treat the disease. AD is currently the third mostexpensive disease after heart disease and cancer. Treatment and care ofthose with AD now runs $100 to $150 billion a year and costs areprojected to rise sharply as the population ages. Currently,pharmacological treatment of AD is primarily based on the use ofacetylcholinesterase inhibitors (AChEIs), which have been reported toprovide beneficial effects on cognitive, functional, and behavioralsymptoms of the disease. Four of the five drugs approved for ADtreatment in the U.S.—donepezil (Aricept®), rivastigmine (Exelon®),galantamine (Reminyl®), and tacrine (Cognex®)—are AChEIs. The fifth,Mementine (Namenda®), is an N-methyl-D-aspartate (“NMDA”) antagonistthat alters glutamate signaling. Because only a small fraction of ADpatients respond to this type of treatment, any new approach to thetreatment or prevention of AD would have tremendous value. In spite ofintensive research, there are no proven preventive agents or agentscapable of limiting progression of the disease, and the few used toameliorate its symptoms have side effects of nausea, vomiting, diarrhea,and even liver damage, yet do not meaningfully slow the disease'sunderlying course for most patients. AD may well be the mostpharmaceutically under-served major disease in the US.

AD is physically evidenced by amyloid plaques and neurofibrillarytangles in the brain. These pathological markers are associated withcognitive regression and other varied symptoms of the disease. Theamyloid plaques, which are the focus of the preponderance of researchtoday on the disease, contain aggregated amyloid β-peptides derived fromproteolytic cleavage of the larger amyloid precursor protein. The majorcomponent of neurofibrillary tangles is the protein tau, a constituentof the cytoskeleton. Tau is a microtubule-associated protein thatfunctions in brain to regulate the structure and function of axonalmicrotubules. Over the past decade, several groups have demonstratedthat the tau protein found in neurofibrillary tangles ishyperphosphorylated. Tau hyperphosphorylation is thought to destabilizemicrotubules and thereby contribute to neurodegenertion and thedevelopment of AD. Tau hyperphosphorylation results from an imbalancebetween kinase and phosphatase activities (reviewed in Buee et al.,Brain Res Brain Res Rev 33(1): 95-130 (2000)). Several serine/threonineprotein kinases have been implicated in tau phosphorylation includingcyclin-dependent kinase 5 (“cdk-5”), glycogen synthase kinase-3b(“GSK-3b”) and MAP kinases. Tau dephosphorylation appears to beprimarily mediated by protein phosphatase 2A (“PP2A”). Importantly,recent results suggest that a decrease in PP2A activity is associatedwith the elevated levels of tau phosphorylation that appear to causeneurofibrillary tangle formation (Planel et al., J Biol Chem.276(36):34298-306 (2001)). Enhancement of this activity therefore mayhave significant therapeutic value. Since PP2A methylation greatlyenhances the formation of a PP2A heterotrimer, it is believed thatenhancing PP2A methylation will result in enhanced PP2A activity towardsTau.

Protein phosphatase 2A (PP2A) associates with a variety of regulatorysubunits. (Janssens, V., Gloris, J., Biochem. J. 353 (Pt. 3): 417-39(2001)). The predominant form in neuronal tissue is a trimer composed ofa dimeric core composed of a 65 kilodalton (kDa) A subunit and the 36kDa PP2A catalytic C subunit associated with one of several differentregulatory B subunits. Whereas the A and C subunits are present more orless uniformly, the B subunit is variable and confers substratespecificity and subcellular localization to each PP2A holoenzyme trimer.The number and types of B subunits present is subject to developmentalregulation and is cell type specific.

The variable B subunits of PP2A are classified into four families: (1)the B family with four isoforms (α, β, γ, δ); (2) the B′ family withfive isoforms (α, β, γ, δ, ε); (3) the B″ family; and (4) the B′″family. The PP2A AB_(α)C heterotrimer appears to be the majorphosphatase in brain responsible for dephosphorylation of tau.(Kamibayashi, C. et al., J. Biol. Chem. 269 (31): 20139-148 (1994);Sontag, E., et al., J. Neuropathol. Exp. Neurol. 63 (4): 287-301(2004)).

The alpha-carboxyl of the C terminal leucine residue of the catalyticsubunit of PP2A is subject to methyl esterification and methyl-esterhydrolysis, and the methylation state of PP2A regulates heterotrimerformation. (Tokstykh, T. et al., EMBO J. 19 (21): 5682-91 (2000); Wu, J.et al., EMBO J. 19 (21): 5672-81 (2000); Wei, H. et al., J. Biol. Chem.276 (2): 1570-77 (2001); Yu, X X, et al., Mol. Biol. Cell 12 (1): 185-99(2001)). Two enzymes are involved in controlling the methylation stateof PP2A: (1) an S-adenosylmethionine-dependent PP2A-specific proteinmethyltransferase (“PPMT”), which adds the methyl group and (2) aPP2A-specific protein methylesterase (“PPME”), which removes the methylgroup. PP2A methylation promotes PP2A AB_(α)C trimer assembly. Anydeficiency in methylation is expected to preclude PP2A AB_(α)Cheterotrimer formation, thereby leading to a deficiency in taudephosphorylation, tau hyperphosphorylation and the formation ofneurofibrillary tangles. (Vafai, S. B., Stock, J. B., FEBS Lett. 518(1-3): 1-4 (2002)).

Homocysteine, a sulfur-containing amino acid that can be eitherremethylated to methionine or undergo a trans-sulfuration reaction tocystathionine, plays a key role in methylation metabolism (see FIG. 1).The conversion of homocysteine to methionine occurs in all tissues.Methionine is activated by ATP in the presence of methionine adenosyltransferase (labeled as (1) in FIG. 1) to form the methyl donor,S-adenosylmethionine (“SAM”). SAM-dependent methylation reactions in thepresence of SAM-dependent methyltransferases (labeled as (2) in FIG. 1)result in the formation of S-adenosylhomocysteine (“SAH”), which iscleaved by SAH hydrolase (labeled as (3) in FIG. 1) to form adenosineand homocysteine. This reaction is reversible with the equilibriumfavoring the condensation of homocysteine and adenosine. Under normalconditions, homocysteine is rapidly methylated, which favors the furthercleavage of SAH. Homocysteine accumulation leads to global decreases incellular methylation by the condensation of homocysteine with adenosineto form SAH, which acts as a product inhibitor in cellular methylationreactions. In the United States, 5-10% of the general population haselevated plasma homocysteine, and this imbalance increases to 30-40% inof the elderly. (Selub J., et al., Ann. Intern. Med. 131 (5): 331-39(1999)). The enzymes cystathionine L-synthase; cystathionine Q lyase;betaine homocysteine methyltransferase; and methionine synthase arelabeled as (4), (5), (6) and (7), respectively in FIG. 1. See Vafai, S.B., Stock, J. B., FEBS Lett. 2: 518 (2002).

Over the last several years, data has emerged in clinical literaturesuggesting a direct association between elevated plasma homocysteine andthe occurrence of AD. Seshadri et al., (N Engl J Med 346 (7): 476-83(2002)), demonstrated that elevated homocysteine is a risk factor forAD. After adjusting for other AD risk factors, the study concluded thatplasma homocysteine levels greater than 14 μM coincided with about a2-fold increased risk for developing AD with an additional 40% increasedrisk with each 5 μM incremental rise. Other diseases, conditions ordisorders associated with elevated plasma homocysteine include, but arenot limited to, atherosclerosis; neurodegenerative disorders, such asParkinson's disease; cerebrovascular disorders (i.e., disorderspertaining to blood vessels in the brain), such as stroke;neuropsychiatric disorders, such as bipolar disorder and schizophrenia;diabetes (type II), and arthritis.

An analysis of the clinical and basic science literature indicates thata methylation defect resulting from elevated homocysteine could lead tolowered levels of PP2A methylation that would result in lowered PP2AAB_(α)C, which is believed to lead to tau hyperphosphorylation,neurofibrillary tangle formation, and dementia (Vafai and Stock, FEBSLett 518(1-3): 1-4 (2002)).

Cellular pathways for removing plasma homocysteine require folate,Vitamin B6 and B12, and thus high homocysteine levels are expected inmice fed diets deficient in these components. This was demonstratedusing, male C57BL/J6 mice. One set of 4 week old mice were placed on adiet that contained folate, vitamin B6, and vitamin B12 and another setwere fed diets that lacked these vitamins. The mice were allowed freeaccess to both food and water. After nine weeks on their respectivediets, each mouse was sacrificed by cervical dislocation. Blood sampleswere collected for measurement of plasma homocysteine and the brain wasremoved and quickly frozen in liquid nitrogen for further analysis oftau phosphorylation. As expected the vitamin-deficient diets causedsubstantial increases in plasma Hcy and brain SAH. These increases wereaccompanied by elevated levels of Tau phosphorylation, as shown in FIG.2 FIG. 2 provides Western blots after SDS-PAGE of extracts prepared fromthe brains of mice raised on normal diets (A and B) andvitamin-deficient diets (C and D). CP13 and PHF1 are monoclonalantibodies that are specific for phosphorylated tau epitopes. TG5 is amonoclonal antibody that recognizes tau independent of its state ofphosphorylation; it thereby provides a control showing that total levelsof tau expression are unaffected by diet. Mice raised on diets deficientin folate, B12, and B6 had dramatically elevated levels of total plasmahomocysteine, brain S-adenosyl homocysteine and elevated levels of tauphosphorylation. S-Adenosyl methionine levels were not significantlyaffected.

The demographics of aging in the United States population, combined witha lack of effective treatments, have heightened the need for ADtherapies. Moreover, the development of preventives would be an evengreater contribution to public health. A protective agent that could betaken over many years to reduce the risk of AD or to substantively delayits onset would be an invaluable breakthrough.

Coffee has been used for centuries by a diverse range of populations andis presently the most popular beverage worldwide with over 400 billioncups consumed each year. There are many anecdotal reports of themedicinal value of coffee but in spite of its worldwide prevalence,little is really known about its potential medical uses. Someepidemiological studies have suggested an inverse association betweencoffee consumption and the risk not only of AD, but also of livercirrhosis, colorectal cancer, cardiovascular mortality, type 2 diabetesand Parkinson's disease. Recent studies have suggested that coffeeconsumption reduces the risk for AD by as much as 30% (Lindsay et al.,Am J Epidemiol. 156(5):445-53 (2002)). Various mechanisms for thepurported benefits have been suggested, but none have been exploredfully enough for these suggestions to be definitive. Moreover, brewedcoffee is a complex mixture that contains several pharmacologicallyactive components, including caffeine.

The present invention provides a definitive mechanism, i.e., enhancementof PP2A methylation, by which to measure benefit to a subject's healthand general well-being, and, in particular, cognitive function. An agentthat increases the levels of PP2A methylation would help maintain highlevels of PP2A ABC heterotrimer formation and may thereby prevent thedemethylation of PP2A and the lowered levels of PP2A ABC heterotrimersthat have been associated with tau hyperphosphorylation, neurofibrillarytangle formation, and neurodegeneration in AD.

SUMMARY OF THE INVENTION

The present invention provides novel compositions that inhibit PP2Ademethylation and thereby prevent negative effects of PP2A demethylationassociated with diseases such as AD and methods to prepare thesecompositions. According to one embodiment of the present invention, acomposition for promoting general health and well-being in a mammaliansubject, including a human, comprises an isolated methylation modifyingcompound. According to another embodiment, the compound of thecomposition is isolated from a botanical raw material. According toanother embodiment, the compound of the composition is isolated from amicrobial raw material. According to another embodiment, the compositioninhibits a demethylating activity of a demethylating enzyme that acts ona protein phosphatase 2A enzyme and thereby stimulates methylation ofthe protein phosphatase 2A enzyme. According to another embodiment, thecomposition inhibits at least about 50% of the demethylating activity ofthe demethylating enzyme. According to another embodiment, thedemethylating enzyme is a protein phosphatase 2A specific proteinmethylesterase. According to another embodiment, the demethylatingactivity of the protein phosphatase 2A specific protein methylesteraseis determined by measuring levels of protein phosphatase 2A methylesterification. According to another embodiment, the botanical rawmaterial comprises at least one substance selected from the groupconsisting of gingko, allicin, bacopa; butcher's broom; flaxseed oil; atocopherol; vitamin E; ginseng, grape seed, St. John's wort;artichoke-powder; choline; inositol; coffee; tea; tobacco; and cocoa.According to another embodiment, the botanical raw material comprises afruit of a species of plant genus Coffea. According to anotherembodiment, the fruit of the plant genus Coffea is a coffee bean.According to another embodiment, the botanical raw material comprises anutritional supplement. According to another embodiment, the nutritionalsupplement comprises at least one tocopherol. According to anotherembodiment, the nutritional supplement comprises Vitamin E. According toanother embodiment, the botanical raw material comprises a leaf of atobacco plant. According to another embodiment, the botanical rawmaterial comprises a ground coffee or an instant coffee. According toanother embodiment, the methylation modifying compound is isolated fromthe botanical raw material by extracting the botanical raw material witha polar solvent. According to another embodiment, the polar solvent is alower alcohol. According to another embodiment, the lower alcohol isethanol. According to another embodiment, the polar solvent is ethylacetate. According to another embodiment, the methylation modifyingcompound is soluble in ethyl acetate below a pH of about 5 and insolublein ethyl acetate above a pH of about 10. According to anotherembodiment, the composition is essentially free of at least onesubstance selected from the group consisting of caffeine, caffeic acidand chlorogenic acid.

According to yet another embodiment of the present invention, acomestible for promoting general health and well-being in a mammaliansubject, including a human, comprises a composition containing anisolated methylation modifying compound. According to anotherembodiment, the compound is isolated from a botanical raw material.According to another embodiment, the compound is isolated from amicrobial raw material. According to another embodiment, the compositioninhibits a demethylating activity of a demethylating enzyme that acts ona protein phosphatase 2A enzyme and thereby stimulates methylation ofthe protein phosphatase 2A enzyme. According to another embodiment, thecomposition inhibits at least about 50% of the demethylating activity ofthe demethylating enzyme. According to another embodiment, thedemethylating enzyme is protein phosphatase 2A specific proteinmethylesterase. According to another embodiment, the level of proteinmethylesterase demethylating activity is determined by measuring levelsof protein phosphatase 2A methyl esterification. According to anotherembodiment, the botanical raw material comprises at least one substanceselected from the group consisting of gingko, allicin, bacopa; butcher'sbroom; flaxseed oil; a tocopherol; vitamin E; ginseng, grape seed, St.John's wort; artichoke-powder; choline; inositol; coffee; tea; tobacco;and cocoa. According to another embodiment, the botanical raw materialcomprises a fruit of a species of plant genus Coffea According toanother embodiment, the fruit of the plant genus Coffea is a coffeebean. According to another embodiment, the botanical raw materialcomprises a nutritional supplement. According to another embodiment, thenutritional supplement comprises at least one tocopherol. According toanother embodiment, the nutritional supplement comprises Vitamin E.According to another embodiment, the botanical raw material comprises aleaf of a tobacco plant. According to another embodiment, the botanicalraw material comprises a ground coffee. According to another embodiment,the botanical raw material comprises an instant coffee. According toanother embodiment, the methylation modifying compound is isolated fromthe botanical raw material by extracting the botanical raw material witha polar solvent. According to another embodiment, the polar solvent is alower alcohol. According to another embodiment, the lower alcohol isethanol. According to another embodiment, the polar solvent is ethylacetate. According to another embodiment, the methylation modifyingcompound is soluble in ethyl acetate below a pH of about 5 and insolublein ethyl acetate above a pH of about 10. According to anotherembodiment, the composition is essentially free of at least onesubstance selected from the group consisting of caffeine, caffeic acidand chlorogenic acid. According to another embodiment, the comestible isa beverage. According to another embodiment, the beverage is selectedfrom the group consisting of a drink comprising water, a fruit drink, acoffee, a tea, an energy drink, a baby formula, an adult nutritionaldrink, a health drink, and a sports drink. According to anotherembodiment, the comestible is a food. According to another embodiment,the comestible is a cereal. According to another embodiment, thecomestible is a chewing gum. According to another embodiment, thecomestible is a candy. According to another embodiment, the comestibleis an ingredient of an inhalant. According to another embodiment, thecomestible is an ingredient of a transdermal delivery system.

According to yet another embodiment of the present invention, apharmaceutical preparation for promoting general health and well-beingin a mammalian subject, including a human, comprises a cognitivefunction-enhancing amount of a composition containing an isolatedmethylation modifying compound and a pharmaceutically acceptablecarrier. According to another embodiment, the compound is isolated froma botanical raw material. According to another embodiment, the compoundis isolated from microbial raw material. According to anotherembodiment, the compound inhibits a demethylating enzyme that acts on aprotein phosphatase 2A and thereby stimulates methylation of the proteinphosphatase 2A enzyme. According to another embodiment, the compositioninhibits at least about 50% of the demethylating activity of thedemethylating enzyme. According to another embodiment, the demethylatingenzyme is a protein phosphatase 2A specific protein methylesterase.According to another embodiment, the demethylating activity of theprotein phosphatase 2A specific protein methylesterase is determined bymeasuring levels of protein phosphatase 2A methyl esterification.According to another embodiment, the botanical raw material comprises atleast one substance selected from the group consisting of gingko;allicin, bacopa; butcher's broom; flaxseed oil; a tocopherol; vitamin E;ginseng, grape seed, St. John's wort; artichoke-powder; choline,inositol; tea; tobacco; and cocoa. According to another embodiment, thebotanical raw material comprises a fruit of a species of plant genusCoffea. According to another embodiment, the fruit of the plant genusCoffea is a coffee bean. According to another embodiment, the botanicalraw material comprises a ground or instant coffee. According to anotherembodiment, the botanical raw material is a nutritional supplement.According to another embodiment, the nutritional supplement comprises atleast one tocopherol. According to another embodiment, the nutritionalsupplement comprises vitamin E. According to another embodiment, thebotanical raw material comprises a leaf of a tobacco plant. According toanother embodiment, the methylation modifying compound is isolated fromthe botanical raw material by extracting the botanical raw material witha polar solvent. According to another embodiment, the polar solvent is alower alcohol. According to another embodiment, the lower alcohol isethanol. According to another embodiment, the polar solvent is ethylacetate. According to another embodiment, the methylation modifyingcompound is soluble in ethyl acetate below a pH of about 5 and insolublein ethyl acetate above a pH of about 10. According to anotherembodiment, the pharmaceutical preparation is essentially free of atleast one substance selected from the group consisting of caffeine,caffeic acid and chlorogenic acid. According to another embodiment, thepreparation is administered orally. According to another embodiment, thepreparation is in the form of a powder. According to another embodiment,the preparation is in the form of a tablet, a capsule, a lozenge, or asuppository.

According to yet another embodiment of the present invention, a methodfor promoting general health and well-being in a mammalian subject,including a human, the method comprises the steps (a) isolating amethylation modifying compound; and (b) administering a cognitivefunction-enhancing amount of a composition comprising the isolatedmethylation modifying compound to a subject in need thereof. Accordingto another embodiment, in step (a), the methylation modifying compoundis isolated from a botanical raw material. According to anotherembodiment, in step (a) the methylation modifying compound is isolatedfrom a microbial raw material. According to another embodiment, themethylation modifying compound inhibits a demethylating enzyme that actson a protein phosphatase 2A enzyme and thereby stimulates methylation ofthe protein phosphatase 2A enzyme. According to another embodiment, thecomposition inhibits at least about 50% of the demethylating activity ofthe demethylating enzyme. According to another embodiment, thedemethylating enzyme is a protein phosphatase 2A specific proteinmethylesterase. According to another embodiment, the method furthercomprises the step of (c) determining the demethylating activity of theprotein phosphatase 2A specific protein methylesterase. According toanother embodiment, the cognition function-enhancing amount of thecomposition is from about 1 mg to about 10 g. According to anotherembodiment, the botanical raw material comprises at least one substanceselected from the group consisting of gingko, allicin, bacopa; butcher'sbroom; flaxseed oil; a tocopherol; vitamin E; ginseng, grape seed, St.John's wort; artichoke-powder; choline; inositol; tea; tobacco; andcocoa. According to another embodiment, the botanical raw materialcomprises a fruit of a species of plant genus Coffea. According toanother embodiment, the fruit of the plant genus Coffea is a coffeebean. According to another embodiment, the botanical raw materialcomprises a ground or instant coffee. According to another embodiment,the botanical raw material comprises a nutritional supplement. Accordingto another embodiment, the nutritional supplement comprises at least onetocopherol. According to another embodiment, the nutritional supplementcomprises vitamin E. According to another embodiment, the botanical rawmaterial comprises a leaf of a tobacco plant. According to anotherembodiment, step (a) further comprises the step of extracting thebotanical raw material with a polar solvent. According to anotherembodiment, the polar solvent is a lower alcohol. According to anotherembodiment, the lower alcohol is ethanol. According to anotherembodiment, the polar solvent is ethyl acetate. According to anotherembodiment, step (a) further comprises the steps of solubilizing themethylation modifying compound in ethyl acetate below a pH of about 5and partitioning the methylation modifying compound between an aqueousphase and ethyl acetate above a pH of about 10. According to anotherembodiment, the extracted composition is essentially free of at leastone substance selected from the group consisting of caffeine, caffeicacid and chlorogenic acid. According to another embodiment, in step (b),the composition is administered orally. According to another embodiment,the composition is a powder, tablet, capsule, lozenge or suppository.

According to yet another embodiment of the present invention, acomposition for promoting general health and well-being in a mammaliansubject, including a human, is prepared by a process, which comprisesthe steps: a) contacting a botanical raw material with a polar solventfor a time sufficient to form an extract solution from the botanical rawmaterial; b) isolating the extract solution and removing the polarorganic solvent to form a concentrated primary extract; and c)dissolving the composition in a solvent at a pH ranging from about 7 toabout 10 to form a concentrated purified extract. According to anotherembodiment, the polar solvent in step (a) of the process is a loweralcohol. According to another embodiment, the lower alcohol is ethanol.According to another embodiment, the polar solvent in step (a) of theprocess is ethyl acetate. According to another embodiment, step (a) ofthe process further comprises the step of solubilizing the compositionin ethyl acetate below a pH of about 5. According to another embodiment,step (b) of the process further comprises the step of partitioning themethylation modifying compound between an aqueous phase and ethylacetate above a pH of about 10. According to another embodiment, thecomposition inhibits a demethylating activity of a demethylating enzymethat acts on a protein phosphatase 2A enzyme and thereby stimulatesmethylation of the protein phosphatase 2A enzyme. According to anotherembodiment, the composition inhibits at least about 50% of thedemethylating activity of the demethylating enzyme. According to anotherembodiment, the demethylating enzyme is a protein phosphatase 2Aspecific protein methylesterase. According to another embodiment, thebotanical raw material comprises at least one substance selected fromthe group consisting of gingko; allicin; bacopa; butcher's broom;flaxseed oil; a tocopherol; vitamin E; ginseng; grape seed; St. John'swort; artichoke-powder; choline; inositol; coffee; tea; tobacco; andcocoa. According to another embodiment, the botanical raw materialcomprises a fruit of a species of plant genus Coffea. According toanother embodiment, the fruit of the plant genus Coffea is a coffeebean. According to another embodiment, the botanical raw materialcomprises a ground or instant coffee. According to another embodiment,the botanical raw material comprises a nutritional supplement. Accordingto another embodiment, the nutritional supplement comprises at least onetocopherol. According to another embodiment, the nutritional supplementcomprises Vitamin E. According to another embodiment, the botanical rawmaterial comprises a leaf of a tobacco plant. According to anotherembodiment, the concentrated purified extract is essentially free of atleast one substance selected from the group consisting of caffeine,caffeic acid and chlorogenic acid. According to another embodiment, thecomposition is administered orally. According to another embodiment, thecomposition is in the form of a powder, a tablet, a capsule, a lozengeor a suppository.

According to yet another embodiment of the present invention, a methodof treating or preventing cognitive effects of a disease, condition, ordisorder involving defective methylation metabolism in a mammaliansubject, including a human, comprises the steps of (a)

isolating a methylation modifying compound; and (b) administering atherapeutically effective amount of a composition comprising theisolated methylation modifying compound to a subject in need thereof.According to another embodiment, in step (a) the methylation modifyingcompound is isolated from a botanical raw material. According to anotherembodiment, in step (a) the methylation modifying compound is isolatedfrom a microbial raw material. According to another embodiment, thedisease, condition, or disorder is associated with higher than normallevels of plasma homocysteine. According to another embodiment, thedisease condition or disorder is at least one disease, condition ordisorder selected from the group consisting of a cardiac disorder;atherosclerosis; a neurodegenerative disorder; a cerebrovasculardisorder; a neuropsychiatric disorder; and diabetes. According toanother embodiment, the methylation modifying compound inhibits ademethylating activity of a demethylating enzyme that acts on a proteinphosphatase 2A enzyme and thereby stimulates methylation of the proteinphosphatase 2A enzyme. According to another embodiment, the compositioninhibits at least about 50% of the demethylating activity of thedemethylating enzyme. According to another embodiment, the demethylatingenzyme is a protein phosphatase 2A specific protein methylesterase.According to another embodiment, the therapeutically effective amount ofthe composition is from about 1 mg to about 10 g of the composition.According to another embodiment, the botanical raw material comprises atleast one substance selected from the group consisting of gingko,allicin, bacopa; butcher's broom; flaxseed oil; a tacopherol; Vitamin E;ginseng, grape seed, St. John's wort; artichoke-powder; choline;inositol; tea; tobacco; and cocoa. According to another embodiment, thebotanical raw material comprises a fruit of a species of plant genusCoffea. According to another embodiment, the fruit of the plant genusCoffea is a coffee bean. According to another embodiment, the botanicalraw material comprises a ground or instant coffee. According to anotherembodiment, the botanical raw material comprises a nutritionalsupplement. According to another embodiment, the nutritional supplementcomprises at least one tocopherol. According to another embodiment, thenutritional supplement comprises Vitamin E. According to anotherembodiment, the botanical raw material comprises a leaf of a tobaccoplant. According to another embodiment, step (a) further comprises thestep of extracting the botanical raw material with a polar solvent.According to another embodiment, the polar solvent is a lower alcohol.According to another embodiment, the polar solvent is ethyl acetate.According to another embodiment, step (a) further comprises the steps ofsolubilizing the methylation modifying compound in ethyl acetate below apH of about 5 and partitioning the methylation modifying compoundbetween an aqueous phase and ethyl acetate above a pH of about 10.According to another embodiment, the composition is essentially free ofat least one substance selected from the group consisting of caffeine,caffeic acid and chlorogenic acid. According to another embodiment, instep (b) the composition is administered orally. According to anotherembodiment, the composition is a powder, a tablet, a capsule, a lozenge,or a suppository.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic diagram of the methyl cycle (1: methionineadenosyl transferase; 2: SAM-dependent methyl transferases; 3: SAHhydrolase; 4: Betaine homocysteine methyl transferases; 5: methioninesynthase).

FIG. 2 provides Western blots after SDS-PAGE of extracts prepared fromthe brains of mice raised on normal diets (A and B) andvitamin-deficient diets (C and D).

FIG. 3 demonstrates that an ethanol extract prepared from coffee groundsshows a concentration dependent inhibition of PP2A methyl esterase.

FIG. 4 parts A through D show purification of PP2A methyl esteraseinhibitor compound activity extracted from instant coffee by ethanolextraction: (A) PP2A methyl esterase inhibitor compound activity incrude ethanol extract after three 100 ml ethanol extractions, 300 mltotal volume; (B) PP2A methyl esterase inhibitor compound activity incrude ethanol extract after two 100 ml and one 50 ml ethanolextractions, 200 ml total volume; (C) PP2A methyl esterase inhibitorcompound activity in water wash compared to inhibitor remaining in thewater insoluble material. (D) elution profile from a C18 solid phaseextraction cartridge showing further purification of PP2A methylesterase inhibitor compound.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses novel compositions extracted frombotanical and microbial extracts, methods of making the compositions,and methods of treating or preventing human disease using the novelcompositions. The inventive extracts can dramatically decrease thedemethylation of PP2A, thus increasing cognitive function and theresilience of cognitive function, particularly in persons suffering fromor prone to developing Alzheimer's disease.

The present invention is based on the recognition that a botanicalextract that stimulates methylation of PP2A or inhibits thedemethylation of PP2A may be useful to treat certain health problems,including, but not limited to AD, that have been associated withdecreases in PP2A methylation. It has long been believed that coffeeproducts, in particular the caffeine in such products, aid in mentalfocus, learning, and memory. In one embodiment of the present invention,an extract of coffee that is essentially free of caffeine is shown topossess a botanical ingredient having PP2A-specific proteinmethylesterase inhibitor activity capable of enhancing cognitivefunction.

Definitions

The term “active constituent” is defined as the chemical constituent ina botanical raw material or a microbial raw material that is responsiblefor the intended therapeutic effect.

“Alkyl” as used herein refers to a straight or branched chain optionallysubstituted hydrocarbon having from one to 10 carbon atoms. Examples of“alkyl” as used herein include, but are not limited to methyl, ethyl,isopropyl n-butyl, t-butyl, and the like.

The term “botanical raw material” as used herein refers to a fresh orprocessed (e.g. cleaned, frozen, dried, sliced, dissolved, or liquefied)part of a single species of plant or a fresh or processed alga ormacroscopic fungus.

The term “botanical ingredient” refers to a component that originatesfrom a botanical raw material.

The term “botanical product” refers to a finished, labeled product thatcontains vegetable matter, which may include plant materials, algae,macroscopic fungi, or combinations thereof. Depending in part on itsintended use, a botanical product may be a food, drug, medical device orcosmetic.

The term “botanical extract” as used herein refers to a product preparedby separating, by chemical or physical process, medicinally activeportions of a plan from the inactive or inert components. The botanicalextracts prepared according to the present invention preferably areobtained by means of a solvent, optionally under pressure and/or heat.

As used herein, the term “cognitive function” refers to the ability toperform mental tasks, such as thinking, learning, judging, remembering,computing, controlling motor functions, and the like. The expression“resilience of cognitive function” refers to the ability of functionalelements of cognitive function to resist deterioration over time. Asused herein, the term “cognitive function enhancing amount” refers tothat amount of the composition of the present invention that willnoticeably impact the ability to perform mental tasks, as measured bytests for memory, computation, attention, or other mental or cognitiveattribute, or as suggested by an individual's perception of his or herabilities in these realms.

As used herein, the term “comestible” refers to a material that issuitable for human consumption, including a material that can beingested by oral and by a non-oral means, e.g., an inhalant or a snuff.For purposes of the present invention, the term includes supplemented orenhanced foods.

The terms “dietary supplement” and “nutritional supplement” are usedinterchangeably herein to mean (1) a product intended to supplement thediet that bears or contains one or more of the following dietaryingredients: [A] a vitamin, [B] a mineral, [C] an herb or otherbotanical, [D] an amino acid, [E] a dietary substance for use by man tosupplement the diet by increasing the total dietary intake; or (F) aconcentrate, metabolite, constituent, extract, or combination of anyingredient described in clause (A), (B), (C), (D), or (E); and (2) aproduct that (A)(i) is intended for ingestion; (B) is not representedfor use as a conventional food or as a sole item of a meal or the diet;and (C) is labeled as a dietary supplement. For purposes of the presentinvention, this definition includes tobacco.

The term “essentially free” means less than about 10% of the amountfound in unprocessed material. For example, if a coffee bean containsabout 1% w/w caffeine, then an extract that is essentially free ofcaffeine would contain less than about 0.1% caffeine w/w (excludingadditional mass due to dilution in water).

The term “extracting” as used herein refers to the process of drawingout, withdrawing, distilling or otherwise separating one substance fromanother by a chemical or physical process.

The term “food” as used herein refers to (a) articles used for food ordrink for man or other animals; (2) chewing gum; and (3) articles usedfor components of any such article.

The term “health” or “healthy” as used herein refers to a generalcondition of the body or mind with reference to soundness and vigor, aswell as freedom from disease or ailment.

The term “lower alcohol” refers to a chemical compound of the generalform R—OH, where R is an alkyl group having between one and six carbons.

As used herein, the phrase “methylation modifying compound” refers to anagent that either directly inhibits at least one enzyme thatdemethylates PP2A, including, but not limited to a PP2A-specific proteinmethylesterase, or that indirectly affects the methylation state of PP2Aitself.

The term “microbe” or “microorganism” are used interchangeably herein torefer to an organism too small to be seen clearly with the naked eye,including, but not limited to, microscopic bacteria, fungi (molds),algae, protozoa, and viruses.

A “microbial raw material” as used herein refers to a fresh or processed(e.g. concentrated, frozen, dried, dissolved, liquefied, pelleted) partof a microbial culture.

The term “microbial ingredient” refers to a component that originatesfrom a microbial raw material.

The term “microbial product” refers to a finished, labeled product thatcontains matter derived from a microbial culture.

The term “partitioning” as used herein refers to a process that takesadvantage of the differential solubility of a substance in two solvents.

The terms “soluble” and “solubility” refer to the property of beingsusceptible to being dissolved in a specified fluid (solvent). The term“insoluble,” as used herein refers to the property of a material thathas minimal or limited solubility in a specified solvent.

The term “well-being” as used herein refers to a subject's physical andmental soundness.

Compositions of the Present Invention

One aspect of the present invention provides compositions comprising amethylation modifying compound isolated from a botanical raw material ormicrobial raw material (i) that inhibits at least one enzyme thatdemethylates PP2A, wherein the composition inhibits at least 50%, morepreferably by at least 90%, of the demethylating activity of thedemethylating enzyme as measured by levels of PP2A methylesterification; or (ii) that stimulates the methylating activity of atleast one enzyme that methylates PP2A. In a preferred embodiment, themethylation modifying compound comprises the portion of a botanicalextract prepared from fruit of a species of the plant genus Coffea thatis insoluble in ethyl acetate above a pH of about 10, yet that issoluble in ethyl acetate below a pH of about 5. Preferably, thecomposition is essentially free of caffeine, caffeic acid and/orchlorogenic acid. Caffeine, also known as trimethylzanthine, caffeine,theine, mateine, guaranine, methyltheobromine and1,3,7-trimethylzanthine, is a xanthine alkaloid found naturally incoffee beans, tea, kola nuts, Yerba mate, guarana berries, and the like.Caffeic acid (3-(3,4-Dihydroxyphenyl)-2-propenoic acid), which istotally unrelated to caffeine, is found in many fruits, vegetables,seasonings and beverages consumed by humans, principally in conjugatedforms such as chlorogenic acid. Chlorogenic acid, an ester of caffeicacid and quinic acid, is a major phenolic compound in coffee, isolatedfrom the leaves and fruits of dicotyledonous plants. Chlorogenic acidalso slows the release of glucose into the bloodstream after a meal. Askilled artisan will appreciate that if desired, caffeine and/orchlorogenic acid may be added to the composition.

In one embodiment, the composition of the invention is prepared byextracting coffee beans, the fruit of the coffee tree, either green,roasted or otherwise treated, of C. arabica, C. robusta, C. liberica, C.arabusta, or other species. The extraction procedure concentrates orisolates those agents in coffee that increase the methylation levels ofPP2A. The composition of the invention includes extracts or compositionsof coffee that are selected, isolated, bred, or genetically modified soas to increase the concentrations of the agents or classes of agentshaving these activities and includes compositions of coffee that arefortified with these agents extracted either from coffee or from othersources.

Our experiments with coffee extracts indicate that coffee containsagents that prevent the demethylation of PP2A by PPME and that thesedemethylation inhibitors are distinct from caffeine or its derivatives.Inhibition of this esterase results in an overall higher level ofmethyl-PP2A, and this would be expected to prevent tauhyperphosphorylation and tangle formation associated with AD. Our assayfor this activity involves incubating the test compound with purifiedPPME and purified ³H-methyl-labeled PP2A in 50 mM MOPS buffer at pH 7.2.We have shown that extracts of several herbal substances have asignificant inhibitory effect on PPME. We have analyzed a drip-brewedcoffee embodiment of the present invention and extracts of coffeeembodiments of the present invention in most detail (See Examples 4, 6,and 7). We have discovered that the inhibitor effect is due to a smallmolecule that is soluble in polar solvents and is not caffeine,chlorogenic acid or caffeic acid.

In another aspect, the present invention provides comestibles comprisinga composition comprising a methylation modifying compound that inhibitsat least one enzyme that demethylates PP2A, wherein the compositioninhibits at least 50%, more preferably at least 90%, of thedemethylating activity of the demethylating enzyme as measured by levelsof PP2A methyl esterification, and wherein the methylation modifyingcompound is extracted from a botanical or microbial extract. Preferablythe composition is extracted from fruit of a species of the plant genusCoffea. The comestibles of the invention include fruit-based drinks,coffee-based drinks, tea-based drinks, sport drinks, nutrition bars,snack foods, gums, cereals, candies, baby formulas, energy drinks, adultnutritional drinks, health drinks, and other food products. The term“sports drink” refers to a beverage that is supposed to rehydrateathletes, as well as restoring electrolytes, sugar and other nutrients,for example, Gatorade, POWERade, and All Sport. As used herein, the term“energy drink” refers to a beverage, including, but not limited to, JoltCola, Red Bull and similar products, that contains legal stimulants,vitamins and minerals; these products are formulated to give the user aburst of energy. The term “adult nutritional drink” as used hereinrefers to such products as Ensure, Longetics® or a similar product. Theterm “health drink” refers to any beverage purported to have beneficialhealth effects, including, but not limited to, reducing inflammation;supporting the immune system; neutralizing infectious agents; preventingclogged arteries, preserving cognitive function and inhibiting cancergrowth. The comestible can comprise an additional ingredient thatconfers cognitive or other health benefits.

Yet another aspect of the invention provides pharmaceutical compositionscomprising a composition comprising a methylation modifying compoundthat inhibits at least one enzyme that demethylates PP2A, wherein thecomposition inhibits at least 50%, more preferably at least 90%, of thedemethylating activity of the demethylating enzyme as measured by levelsof PP2A methyl esterification, and wherein the methylation modifyingcompound is extracted from a botanical or microbial extract. Preferably,the composition is extracted from fruit of a species of the plant genusCoffea. The pharmaceutical composition can be formulated for oralconsumption or in the form of a suppository. The pharmaceuticalcompositions of the invention include powders, tablets, capsules andlozenges.

Another aspect of the invention provides methods of enhancing cognitivefunction in a human, the method comprising the step of administering acognitive function enhancing amount of a composition comprising amethylation modifying compound that inhibits at least one enzyme thatdemethylates PP2A, wherein the composition inhibits at least 50% of thedemethylating activity of the demethylating enzyme as measured by levelsof PP2A methyl esterification, and wherein the methylation modifyingcompound is extracted from a botanical or microbial extract. Preferablythe composition is extracted from fruit of a species of the plant genusCoffea. Alternatively, a method of enhancing cognitive function in ahuman according to the present invention comprising the step ofadministering a comestible comprising a cognitive function enhancingamount of a composition comprising a methylation modifying compound thatinhibits at least one enzyme that demethylates PP2A, wherein thecomposition inhibits at least 50% of the demethylating activity of thedemethylating enzyme as measured by levels of PP2A methylesterification, and wherein the methylation modifying compound isextracted from a botanical or microbial extract.

The invention also provides methods of enhancing memory in a human,which method comprises administering a memory enhancing amount of acomposition or a comestible comprising methods of enhancing cognitivefunction in a human, the method comprising the step of administering acognitive function enhancing amount of a composition comprising amethylation modifying compound that inhibits at least one enzyme thatdemethylates PP2A, wherein the composition inhibits at least 50% of thedemethylating activity of the demethylating enzyme as measured by levelsof PP2A methyl esterification, and wherein the methylation modifyingcompound is extracted from either a botanical or a microbial extract.According to the present invention, the composition can be used inmethods of treating or preventing any disease, condition or disorderwhere defects in methylation metabolism appear to play a role asevidenced by an association of the disease, condition or disorder withplasma homocysteine levels that are elevated relative to normal plasmahomocysteine levels. Such diseases, conditions or disorders include, butare not limited to, neurodegenerative diseases, disorders or conditions,such as Parkinson's disease, neuropsychiatric diseases, disorders orconditions, such as bipolar disorder, Alzheimer's disease, heartdisease, arthritis, diabetes and certain cancers. The term“neurodegenerative” as used herein refers to a disease, condition ordisorder marked by the loss or diminution of an original nerve cellfunction, and the term “neuropsychiatric” relates to organic andfunctional diseases, conditions or disorders of the nervous system.

Yet another aspect of the invention provides processes for preparing acomposition comprising a methylation modifying compound that inhibits atleast one enzyme that demethylates PP2A, wherein the compositioninhibits at least 50%, more preferably at least 90%, of thedemethylating activity of the demethylating enzyme as measured by levelsof PP2A methyl esterification, and wherein the methylation modifyingcompound is extracted from either a botanical extract or a microbialextract. When the composition of the present invention is extracted fromthe fruit of a species of the plant genus Coffea, the process of thepresent invention comprises the steps: determining when the compositioninhibits at least 50%, more preferably at least 90%, of thedemethylating activity of the demethylating enzyme as measured by levelsof PP2A methyl esterification, and optionally treating the fruit toremove caffeine. The extract can be treated further to removechlorogenic acid and/or caffeic acid.

Methods of Preparing Compositions of the Invention

Another aspect of the invention provides a method of preparing aconcentrated purified extract comprising a composition useful forenhancing or maintaining cognitive health. One method of preparing acomposition of the invention is to extract the active agents frombotanical raw material or microbial raw material into organic solventsfrom aqueous solutions at acid or neutral pH. Further concentration ofthe agents can be effected by extracting them from organic solvents backinto aqueous solvents at basic pH. In one preferred embodiment involvinga botanical raw material, the method comprises the steps: (a) contactingthe fruit of a species of plant with a polar organic solvent for a timesufficient to form an extract solution, b) removing particulate matterfrom the extract solution; (c) isolating the extract solution andremoving the polar organic solvent to form a concentrated primaryextract, (d) washing the concentrated primary extract with a solvent inwhich impurities are soluble and the agent is poorly soluble; (e)removing the solvent; and (f) dissolving the desired methylationmodifying agent in a polar organic solvent or in water at neutral tobasic pH. For example, a coffee extract is prepared by:

a) contacting a species of the fruit of a species Coffea with pureethanol at an elevated temperature (80 C) for about 5-10 minutes, i.e.,a time sufficient to form an ethanol extract solution from the fruit.

b) removing particulate matter from the ethanol extract solution byfiltration or centrifugation;

c) isolating the ethanol extract solution and removing the ethanol byevaporation in a glass flask to form a concentrated primary extract;

d) washing the concentrated extract with deionized water at a pH belowabout 5;

e) drying the extract to remove residual water; and

f) dissolving the extract in ethanol to form a washed concentratedprimary extract.

The term “solvent” as used herein refers to a substance, usually liquid,capable of dissolving or dispersing one or more other substances.Chemists have classified solvents into two broad categories according totheir polarity: polar and nonpolar. A common measure of the polarity ofa solvent is the dielectric constant. The term “polar solvent” as usedherein refers to a compound that is composed of polar molecules. A“polar molecule” is one in which there is some separation of charge inthe chemical bonds, so that one part of the molecule has a slightpositive charge and the other a slight negative charge. Polar solventsmay be further classified as protic or aprotic. The term “protic” refersto a hydrogen atom attached to an electronegative atom, while the term“aprotic” refers to a molecule that does not contain an O—H bond. A“polar protic solvent” can be represented by the general formula ROH;the polarity of the polar protic solvent stems from the bond dipole ofthe O—H bond. Examples of polar protic solvents include, but are notlimited to, water, alcohols, and acetic acid. A “dipolar aproticsolvent” is one that contains a bond that has a large bond dipole.Typically, this bond is a multiple bond between carbon and either oxygenor nitrogen. Most dipolar aprotic solvents contain a C—O double bond.Examples of dipolar aprotic solvents include, but are not limited to,acetone and ethyl acetate. As the number of —CH₂— groups in ROHincreases, and the relative amount of hydrocarbon character increases,the polarity decreases. The term “nonpolar solvent” refers to compoundsthat have low dielectric constants and are not miscible with water.Examples of nonpolar solvents include, but are not limited to benzene,carbon tetrachloride, and diethyl ether.

Optionally, the washed concentrated primary extract can be dissolved inwater and the pH of the washed concentrated primary extract neutralized.The extract then can be further purified by utilizing the property thatthe desired agent is insoluble in ethyl acetate above a pH of about10.0, and is soluble in ethyl acetate below a pH of about 5.0. It wouldbe apparent to a skilled artisan that by assaying for the desiredeffects on PP2A methylation (see PCT/US03/07658; the contents of whichare incorporated by reference) one can readily identify growthconditions and plant varieties having increased levels of the agents ofthe compositions of the present invention. Likewise, using methods knownin the art, a skilled artisan could genetically engineer or breed plantvarietals to express increased amounts of the desired agents.

The compositions of the present invention may be in a form suitable fororal use, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, solutions, dispersible powders or granules, emulsions, hardor soft capsules, syrups or elixirs, pastes, gels or the like.Compositions intended for oral use may be prepared according to anyknown method, and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents, and preserving agents in order to providepharmaceutically elegant and palatable compositions. Tablets may containthe active ingredient(s) in admixture with non-toxicpharmaceutically-acceptable excipients which are suitable for themanufacture of tablets. These excipients may be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch or alginic acid; binding agents, for example,starch, gelatin or acacia; and lubricating agents, for example,magnesium stearate, stearic acid or talc. The tablets may be uncoated orthey may be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate may be employed. They alsomay be coated for controlled delivery. For example, a “delayed release”dosage form releases a product or substance at a time other thanpromptly after administration. Examples of delayed-release systemsinclude repeat-action tablets and capsules, and enteric-coated tabletswhere timed release is achieved by a barrier coating.

Compositions of the present invention also may be formulated for oraluse as hard gelatin capsules, where the active ingredient(s) is(are)mixed with an inert solid diluent, for example, calcium carbonate,calcium phosphate or kaolin, or soft gelatin capsules wherein the activeingredient(s) is (are) mixed with water or an oil medium, for example,peanut oil, liquid paraffin, or olive oil.

The compositions of the present invention may be formulated as aqueoussuspensions wherein the active ingredient(s) is (are) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatidesuch as lecithin, or condensation products of an alkylene oxide withfatty acids, for example, polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample, heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions also may contain one or more coloring agents,one or more flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Compositions of the present invention may be formulated as oilysuspensions by suspending the active ingredient in a vegetable oil, forexample arachis oil, olive oil, sesame oil or coconut oil, or in amineral oil, such as liquid paraffin. The oily suspensions may contain athickening agent, for example, beeswax, hard paraffin or cetyl alcohol.Sweetening agents, such as those set forth above, and flavoring agentsmay be added to provide a palatable oral composition. These compositionsmay be preserved by the addition of an antioxidant such as ascorbicacid.

Compositions of the present invention may be formulated in the form ofdispersible powders and granules suitable for composition of an aqueoussuspension by the addition of water. The active ingredient in suchpowders and granules is provided in admixture with a dispersing orwetting agent, suspending agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose already mentioned above. Additional excipients, or example,sweetening, flavoring and coloring agents also may be present.

The compositions of the invention also may be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample, olive oil or arachis oil, or a mineral oil, for example aliquid paraffin, or a mixture thereof. Suitable emulsifying agents maybe naturally-occurring gums, for example, gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate. The emulsions also may contain sweetening andflavoring agents.

The compositions of the invention also may be formulated as syrups andelixirs. Syrups and elixirs may be formulated with sweetening agents,for example, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations also may contain a demulcent, a preservative, and flavoringand coloring agents. Demulcents are protective agents employed primarilyto alleviate irritation, particularly mucous membranes or abradedtissues. A number of chemical substances possess demulcent properties.These substances include the alginates, mucilages, gums, dextrins,starches, certain sugars, and polymeric polyhydric glycols. Othersinclude acacia, agar, benzoin, carbomer, gelatin, glycerin, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,propylene glycol, sodium alginate, tragacanth, hydrogels and the like.

The compositions of the present invention also may be in the form of asterile injectable aqueous or oleaginous suspension. Injectablecompositions, such as sterile injectable aqueous or oleaginoussuspensions, may be formulated according to the known art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable composition may also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol. Among the acceptable vehiclesand solvents that may be employed are water, Ringer's solution, andisotonic sodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. Forparenteral application, “parenteral” meaning subcutaneous injections,intravenous, intramuscular, intrasternal injection, or infusiontechniques, particularly suitable vehicles consist of solutions,preferably oily or aqueous solutions, as well as suspensions, emulsions,or implants. Aqueous suspensions may contain substances which increasethe viscosity of the suspension and include, for example, sodiumcarboxymethyl cellulose, sorbitol and/or dextran. Optionally, thesuspension may also contain stabilizers.

The terms “drug carrier”, “carrier”, or “vehicle” are usedinterchangeably to refer to carrier materials suitable foradministration of the methylation modifying compounds. Carriers andvehicles useful herein include any such materials known in the art whichare nontoxic and do not interact with other components. As used hereinthe term “a pharmaceutically acceptable carrier” refers to anysubstantially non-toxic carrier conventionally useable foradministration in which the compound will remain stable andbioavailable.

The compositions of the present invention may be in the form ofsuppositories for rectal administration of the composition. Thesecompositions can be prepared by mixing the active ingredient with asuitable nonirritating excipient such as cocoa butter and polyethyleneglycols which are solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum and release thedrug. When formulated as a suppository the compositions of the inventionmay be formulated with traditional binders and carriers, such astriglycerides.

Compositions of the present invention optionally can include anexcipient. The term “excipients” as used herein refers topharmaceutically acceptable organic or inorganic carrier substanceswhich do not deleteriously react with the active compounds. Suitabledietary excipients include, but are not limited to, dietary suitablestarch, vegetable oil, vegetable gums, gelatins, soy extracts, sugars,grains, natural and artificial flavorings, and the like. Suitablepharmaceutically acceptable carriers include, but are not limited to,water, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil; fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethylcellulose,polyvinylpyrrolidone, and the like. Suitable excipients are described indetail in Remington's Pharmaceutical Sciences, Twentieth Edition, © 2000incorporated herein by reference.

Pharmaceutical compositions can be sterilized and if desired, mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,colorings, flavoring and/or aromatic substances and the like which donot deleteriously react with the active compounds.

The therapeutically active agent of the present invention can beformulated per se or in salt form. The term “pharmaceutically acceptablesalts” refers to nontoxic salts of the active agent. Pharmaceuticallyacceptable salts include, but are not limited to, those formed with freeamino groups such as those derived from hydrochloric, phosphoric,sulfuric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, and the like.

Additional compositions of the present invention can be readily preparedusing technology which is known in the art such as described inRemington's Pharmaceutical Sciences.

A composition of the present invention, alone or in combination withother active ingredients, may be administered to a subject in a singledose or multiple doses over a period of time, generally by oraladministration. As used herein, the terms “therapeutically effectiveamount,” “memory-enhancing amount”, and “cognition enhancing amount” areused interchangeably to refer to the amount of the composition of theinvention that results in a therapeutic or beneficial effect, includinga subject's perception of health or general well-being, following itsadministration to a subject.

It is believed that an increase in the level of PP2A methylation willbring about the protection or enhancement of cognitive functioning, orpreventing a cognitive disorder from manifesting or deepening. Thus thetherapeutic effect of the compositions of the present invention canexert a protective or enhancing effect on cognitive function; minimize,prevent or ameliorate cognitive symptoms of a disease or disorder, ormay have any other beneficial effect.

The concentration of the substance is selected so as to exert itstherapeutic effect, but low enough to avoid significant side effectswithin the scope and sound judgment of the skilled artisan. Theeffective amount of the composition may vary with the age and physicalcondition of the biological subject being treated, the severity of thecondition, the duration of the treatment, the nature of concurrenttherapy, the specific compound, composition or other active ingredientemployed, the particular carrier utilized, and like factors. Those ofskill in the art can readily evaluate such factors and, based on thisinformation, determine the particular effective concentration of acomposition of the present invention to be used for an intended purpose.

A skilled artisan can determine a therapeutically effective amount ofthe inventive compositions by determining the unit dose. As used herein,a “unit dose” refers to the amount of inventive composition required toproduce a response of 50% of maximal effect (i.e. ED₅₀). The unit dosecan be assessed by extrapolating from dose-response curves derived fromin vitro or animal model test systems. The amount of compounds in thecompositions of the present invention which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. (See, for example, Goodman and Gilman's THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, Joel G. Harman, Lee E. Limbird,Eds.; McGraw Hill, New York, 2001; THE PHYSICIAN'S DESK REFERENCE,Medical Economics Company, Inc., Oradell, N.J., 1995; and DRUG FACTS ANDCOMPARISONS, FACTS AND COMPARISONS, INC., St. Louis, Mo., 1993). Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Various administration patterns will beapparent to those skilled in the art.

The dosage ranges for the administration of the compositions of thepresent invention are those large enough to produce the desiredtherapeutic effect. Preferably, the cognitive function enhancing amountof the compositions of the present invention is administered one or moretimes per day on a regular basis. A typical dose administered to a humanis between about 1 mg and about 10 g of the composition, preferablybetween 1 mg and 1 g of the composition.

Those skilled in the art will recognize that initial indications of theappropriate therapeutic dosage of the compositions of the invention canbe determined in in vitro and in vivo animal model systems, and in humanclinical trials. One of skill in the art would know to use animalstudies and human experience to identify a dosage that can safely beadministered without generating toxicity or other side effects. Foracute treatment where it is desirable to substantially increasemethylated PP2A, it is preferred that the therapeutic dosage be close tothe maximum tolerated dose. For chronic preventive use, lower dosagesmay be desirable because of concerns about long term effects. However,coffee and coffee extracts are commonly believed to be safe and have ahistory of human use. The composition preferably is administered one ormore times per day, in food or beverage, as inhalant, in a gum,transdermally, as a suppository, or as a snuff. As used herein, the term“inhalants” refers to substances or combinations of substances that, byvirtue of their high vapor pressure, can be carried by an air currentinto the nasal passage where they exert their effect. The container fromwhich the inhalant is administered is “an inhaler.”

Alternatively, the composition of the present invention may beadministered at least once per day in combination with a prescribeddrug. For example, the composition of the present invention may beadministered together with existing anti-cholinesterase drugs nowprescribed for Alzheimer's, with various anti-inflammatory agents, orwith statins.

In another aspect, the composition of the present invention isadministered at least once per day in combination with a dietary ornutritional supplement believed to have beneficial health effects. Forexample,

Coenzyme Q₁₀ (also known as CoQ₁₀, Q₁₀, vitamin Q₁₀, ubiquinone andubidecarenone), a benzoquinone compound synthesized naturally by thehuman body, is used by cells of the body in oxidative metabolism or cellrespiration and as an endogenous antioxidant. An “antioxidant” is asubstance that protects cells from free radicals, which are highlyreactive chemicals often containing oxygen atoms, that are capable ofdamaging important cellular components, such as DNA and lipids. Theplasma level of CoQ₁₀ has been used in studies as a measure of oxidativestress, a situation in which normal antioxidant levels are reduced.Various investigations have explored the usefulness of CoQ₁₀ as atreatment for diseases, including, but not limited to, cancer andcardiovascular disease.

Idebenone, a synthetic analog of CoQ₁₀, has been investigated in elderlypatients with dementia. Studies suggest that it may diminish nerve celldamage due to ischemia and facilitate memory and learning.

Huperzine A, a natural acetylcholinesterase inhibitor derived from theChinese herb Huperzia serrata, has antioxidant and neuroprotectiveproperties, and has been proposed as a disease-modifying treatment forAD.

Galantamine, an acetylcholinesterase inhibitor, is used to treatsymptoms of AD.

Vincamine and vinpocetine, a semisynthetic derivative of vincamine, analkaloid derived from the plant Vina minor L, are used in Europe, Japanand Mexico as pharmaceutical agents for the treatment of cerebrovascularand cognitive disorders.

Acetyl-L-carinitine, an acetylated derivative of carnitine, has beenshown to promote fatty acid beta-oxidation in liver and to prevent motornerve condition velocity slowing in diabetic rats.

Dehydroepiandrosterone (DHEA), a steroid, is being studied in theprevention of cancer. In the body, it is a precursor produced by theadrenal gland and converted to testosterone or the estrogens.

Phosphatidylcholine, a phospholipid that is a major component of cellmembranes, has putative activity as a cognition enhancer and incell-membrane repair

Gingko, an herb, has putative properties as a neuroprotective agent, anantioxidant, a free-radical scavenger, a membrane stabilizer, and aninhibitor of platelet-activating factor. Sherpina, V. S., et al.,American Family Physician 68(5) 923-926 (2003). Gingko extract also hasbeen shown to inhibit beta-amyloid deposition. Id.

Circumin, an active ingredient in turmeric, which is in curry,purportedly has anti-inflammatory and cholesterol lowering properties.

Ginseng, a Chinese herb, has been used for centuries in Asia as a curefor many maladies.

Research has shown that Vitamin E (DL-alpha-tocopherol), an essentialvitamin that functions as an antioxidant, can help preventcardiovascular disease and increase the immune response. It has beenhypothesized that Vitamin E and its analogs and derivatives may preventbrain cell damage by destroying toxic free radicals. The term “tocol”generally refers to 2-methyl-2-(4,8,12-trimetyltridecyl)chroman-6-ol;the term “tocopherol” generally refers to all mono, di, andtrimethyltocols, including, but not limited to, alpha-tocopherol(5,7,8-trimethyltocol), beta-tocopherol (5,8-dimethyltocol),gamma-tocopherol (7,8-dimethyltocol), delta-tocopherol (8-methyltocol),the term “tocotrienol” refers to2-methyl-2-(4,8,12-trimethyltrideca-3,7,11-trienyl)chroman-6-ol; and theterm “vitamin E” generally refers to all tocol and tocotrienolderivatives exhibiting qualitatively the biological activity ofalpha-tocopherol.

It is well-known that N-acetyl-cysteine (NAC) promotes cellularglutathione production, and thus reduces, or even prevents, oxidantmediated damage. Treatment with NAC provides beneficial effects in anumber of respiratory, cardiovascular, endocrine, infectious, and otherdisease settings.

B vitamins, such as folic acid, are known to reduce levels ofhomocysteine, an amino acid already linked, at high levels, to anincreased risk of heart attacks, strokes and Alzheimer's disease.

Lecithin, a lipid material composed of choline and inositol, is a majorcomponent of cell membranes. As used by producers of lecithin forcommercial use, the term “lecithin” refers to a complex mix ofphosphatides and other substances that contain phosphatidylcholine.

Choline (trimethyl ethanolamine), a quaternary saturated amineclassified as an essential nutrient by the Food and Nutrition Board ofthe Institute of Medicine, is a component of lecithin. Choline is neededby the body to make the neurotransmitter acetylcholine.

Fish oil, which is oil derived from the tissues of oily fish, naturallycontains the omega-3 fatty acids eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA). Some experts believe that fish oil can helpregulate cholesterol in the body. It also may help protect the brainfrom cognitive problems associated with Alzheimer's disease.

Deprenyl (selegiline, Eldepryl®), a monoamine oxidase inhibitor, isprescribed for the treatment of early-stage Parkinson's disease andsenile dementia.

The compositions of the invention can be used alone or in combinationwith other pharmaceuticals or herbals to prolong mental health, tomaintain or enhance cognitive functioning or memory, or to preservemental or physical well-being and health. The compositions can also beused to prevent or treat effects a number of ailments, including, butnot limited to, Alzheimer's disease; Parkinson's disease; heart disease;arthritis; age-related degeneration, functional impairments, anddiseases; diabetes, and cancer, have on cognitive function.

The effectiveness of the compositions and methods of the presentinvention can be assayed by a variety of protocols. The effects ofincreasing cognitive function in a human subject can be determined bymethods routine to those skilled in the art including, but not limitedto, both paper and pencil, and computer tests. One of skill in the artcan also directly measure PP2A methylation levels, tau proteinphosphorylation levels, neurofibrillary tangle formation andneurodegeneration in animal models.

Comestibles of the Invention

Compositions of the present invention may be included in a variety offorms, including, but not limited to, nutritional supplements,pharmaceutical compositions, vitamin supplements, food additives orfoods supplements. For example, the methylation modifying compoundsdescribed may be embodied as a lozenge, candy, drink, gum, or othersnack, food, pill, snuff, nutritional or food supplement, or deliveredas an ingredient of an inhalant, beverage, or transdermal deliverysystem. For example, the methylation modifying compounds of the presentinvention may be added directly to a liquid beverage, such as water.

The compositions of the present invention may be in the form of adispersible dry powder for pulmonary delivery. Dry powder compositionsmay be prepared by processes known in the art, such as lyophilizationand jet milling, as disclosed in International Patent Publication No. WO91/16038 and as disclosed in U.S. Pat. No. 6,921,527, the disclosures ofwhich are incorporated by reference. The composition of the presentinvention is placed within a suitable dosage receptacle in an amountsufficient to provide a subject with a unit dosage treatment. The dosagereceptacle is one that fits within a suitable inhalation device to allowfor the aerosolization of the dry powder composition by dispersion intoa gas stream to form an aerosol and then capturing the aerosol soproduced in a chamber having a mouthpiece attached for subsequentinhalation by a subject in need of treatment. Such a dosage receptacleincludes any container enclosing the composition known in the art suchas gelatin or plastic capsules with a removable portion that allows astream of gas (e.g., air) to be directed into the container to dispersethe dry powder composition. Such containers are exemplified by thoseshown in U.S. Pat. No. 4,227,522; U.S. Pat. No. 4,192,309; and U.S. Pat.No. 4,105,027. Suitable containers also include those used inconjunction with Glaxo's Ventolin® Rotohaler brand powder inhaler orFison's Spinhaler® brand powder inhaler. Another suitable unit-dosecontainer which provides a superior moisture barrier is formed from analuminum foil plastic laminate. The pharmaceutical-based powder isfilled by weight or by volume into the depression in the formable foiland hermetically sealed with a covering foil-plastic laminate. Such acontainer for use with a powder inhalation device is described in U.S.Pat. No. 4,778,054 and is used with Glaxo's Diskhaler® (U.S. Pat. Nos.4,627,432; 4,811,731; and 5,035,237). All of these references areincorporated herein by reference.

The terms “transdermal delivery system”, “transdermal patch”, or “patch”refer to an adhesive system placed on the skin to deliver a timereleased dose of a drug(s) by passage from the dosage form through theskin to be available for distribution via the systemic circulation.Transdermal patches are a well-accepted technology used to deliver awide variety of pharmaceuticals, including, but not limited to,scopolamine for motion sickness, nitroglycerin for treatment of anginapectoris, clonidine for hypertension, estradiol for post-menopausalindications, and nicotine for smoking cessation. Patches suitable foruse in the present invention include, but are not limited to, (1) thematrix patch; (2) the reservoir patch; (3) the multi-laminatedrug-in-adhesive patch; and (4) the monolithic drug-in-adhesive patch;TRANSDERMAL AND TOPICAL DRUG DELIVERY SYSTEMS, pp. 249-297 (Tapash K.Ghosh et al. eds., 1997), hereby incorporated herein by reference. Thesepatches are well known in the art and generally available commercially.

In preferred embodiments, the comestibles of the invention include acoffee without caffeine, a decaffeinated coffee, a coffee-like beverage,a ready-to-drink coffee, a coffee dessert or any other coffee productenriched for, or selected for, PP2A methylation activity and used forthe health functions described above.

When provided to non-human mammalian subjects, the compositions of thepresent invention may be administered separately or may be combined withordinary feed or liquid nourishment to effect the alterations in bodycomposition as described herein.

The compositions of the invention can be combined with ordinary foods toenhance the value of the palatability of the composition. For example,the compositions can be mixed with soft drinks, food supplements, candy,or high-energy bars, and virtually any other food that can besupplemented with a powder or liquid. Thus, the invention specificallyincludes food substances of specific types combined with the compositionof the invention in specified forms and quantities.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Composition of Pure PP2A Methyltransferase

This example provides a method of preparing pure PPMT for use in assaysof demethylation of PP2A. PPMT was obtained using a bacterialoverexpression system consisting of the pMT and pBADESL plasmids inEscherichia coli strain C41(DE3) (obtained from S. Djordjevic,University College London) (George, R. R. et al., Protein Expr. Purif.26(2): 266-74 (2002). LB flasks containing 0.2% (w/v) arabinose wereinoculated and grown at 37° C. to a density of A₆₀₀=0.5. 1 mMisopropyl-b-D-thiogalactoside (IPTG), an artificial inducer of the Lacoperon, was added to induce methyltransferase expression and after 3 hrthe culture was centrifuged and the pellet washed with 0.1 M potassiumphosphate, pH 7. The pellet was resuspended in 50 mM Tris, pH 7,containing 300 mM NaCl, 10% (v/v) glycerol, 10 mM 2-mercaptoethanol,sonicated on an ice/NaCl bath, and centrifuged. The supernatant wasloaded on a pre-equilibrated 10 ml Ni-NTA superflow column, and elutedwith a 150 ml gradient from 0 to 300 mM imidazole. The methyltransferasecontaining fractions were collected, pooled, concentrated and loadedonto a Sephacryl S-100 gel filtration column, eluted, and fractionscollected. The presence of PPMT in the fractions was confirmed bySDS-PAGE analysis. Relevant fractions were pooled and loaded onto apre-equilibrated Toyopearl Super-Q 650S column. The column then waswashed, and the proteins eluted with a gradient of 0 to 500 mM NaCl.Fractions containing pure PPMT were pooled, dialyzed against 50 mM4-morphonolinepropanesulfonic acid, 3-(N-Morpholino)-propanesulfonicacid (MOPS) buffer containing 10% (v/v) glycerol, 1 mM dithiothreitol(DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), and stored at −20°C. with 40% glycerol. The protocol yielded approximately 10 mg of purePPMT per liter of culture media.

Example 2 Pure PP2A Methylesterase

This example provides a method of preparing pure PPME for use in assaysof PP2A methyl esterification. A PP2A methylesterase plasmid (obtainedfrom S. Djordjevic, University College London) was transformed into E.coli strain BL21(DE3) cells. LB flasks were inoculated with culturesgrown overnight and allowed to grow at 37° C. When the culture reached adensity of A₆₀₀=0.5. 1 mM IPTG was added to induce esterase expression.After 3 hr the cultures were centrifuged and the pellet was washed with0.1 M potassium phosphate, pH 7, and resuspended in 50 mM Tris buffercontaining 300 mM NaCl, 10% glycerol, 10 mM 2-mercaptoethanol. Cellswere then disrupted by sonication and the supernatant was loaded on apre-equilibrated 10 ml Ni: NTA superflow column. The column was washedwith Tris-buffer and proteins were eluted with a 150 ml gradient from 0to 300 mM imidazole. The methylesterase containing fractions werecollected, pooled and dialyzed against 50 mM Tris buffer containing 5%(w/v) glycerol and 1 mM DTT. The proteins were then loaded onto apre-equilibrated Toyopearl Super-Q 650S column. The column was washedand proteins collected by applying a gradient from 0 to 500 mM NaCl.Fractions containing pure PPME were pooled, dialyzed against 50 mM MOPSbuffer containing 10% (v/v) glycerol, 1 mM DTT, 1 mM EDTA and stored at−20° C. with 40% glycerol. This protocol gives approximately 10 mg ofpure PPME per liter of culture media.

Example 3 Purification of PP2A AC Dimers

This example provides a method of preparing pure PP2A AC dimers for usein assays of PP2A methyl esterification. (Lee, J., Stock, J., J. Biol.Chem. 268 (26) 19192-195 (1993); Tolstykh, T. et al., EMBO J. 19 (21):5682-91 (2000)). Two frozen bovine brains were suspended into 800 ml ofBuffer A (20 mM MOPS-Na, pH 7.2, 1.0 mM EDTA/DTT and 0.5 mg/L ofaprotinin, leupeptin and pepstatin (“protease inhibitors”), and blendeduntil smooth at 4 C. 25% (NH₄)₂SO₄ was added to the brains, stirred for30 min and centrifuged. (NH₄)₂SO₄ was added to the supernatant to 70%,stirred for 60 min, and centrifuged. The precipitate in the pellet wascollected and dissolved in 200 ml Buffer A and then dialyzed at 4 Cagainst three, 4 liter changes of Buffer A. The dialyzed solution wasclarified by centrifugation and the supernatant was loaded onto aDEAE-Toyopearl 650M column pre-equilibrated with Buffer A. The columnwas washed and PP2A eluted with Buffer A+0.3 M NaCl. The eluted PP2A wasconcentrated in 60 ml Buffer B (50 mM MOPS, pH 7.2, 1 mM EDTA/DTT, 0.5mg/ml protease inhibitors) with 0.8M (NH₄)₂SO₄, loaded onto a TSK phenylcolumn and washed in this buffer before elution with a 1.0 L lineargradient from 0.8M (NH₄)₂SO₄ to 20% ethylene glycol in Buffer B.Fractions with phosphatase activity were collected and concentrated witha Centriprep concentrator before being loaded on a Sephacryl S-200column pre-equilibrated in Buffer B with 0.2M NaCl. Fractions withphosphatase activity were pooled, buffer exchanged, and concentrated to2 ml in Buffer C (50 mM MOPS, pH 7.2, 1 mM EDTA, 1 mM DTT) using aCentriprep concentrator. Concentrated protein was loaded on a Source-15QHPLC column. A gradient from 0.2M to 0.35 M NaCl in Buffer C in 30minutes, then from 0.35 M NaCl to 0.5 M NaCl in Buffer C in 10 minuteswas used to elute the PP2A. Fractions containing the pure PP2A werecollected, concentrated using a Centriprep concentrator, and stored at−20° C. with 40% glycerol.

Example 4 Assay for Methylesterase Inhibition

This example provides a method by which the rate of demethylation ofmethyl PP2A can be determined. For the composition of ³H-labeledmethyl-PP2A, 100 μl reaction mixtures consisting of purified PP2A ACdimers (prepared as described in Example 3), purified PPMT (prepared asdescribed in Example 1), 50 mM MOPS buffer pH 7.2, 5 mM DTT, 1 mM EDTA,1 mg/ml BSA and 10 μl 3H-SAM were incubated at 37° C. for 30 min andloaded onto a desalting column pre-equilibrated in 50 mM MOPS-Na pH 7.2,1 mM DTT, 1 mM EDTA, 200 mM NaCl and 5% glycerol. The sample was elutedwith the same buffer, 50 μl fractions were collected, and each aliquotanalyzed by scintillation counting. Fractions containing ³H-methyl PP2Awere collected and stored at 4° C.

The following assay (see Lee, J. et al., Proc. Nat'l Acad. Sci. U.S.A.93(12): 603-47 (1996)) is referred to subsequently as the “filter papermethod”. The test compound was incubated with purified PPME (prepared asdescribed in Example 2) and purified ³H-methyl-labeled PP2A (thepreparation of which is described in the preceding paragraph) in BufferC (50 mM MOPS, 1 mM EDTA, 1 mM DTT buffer at pH 7.2, as in Example 3).After incubation at 37° C. for a fixed time (typically 10 minutes), thereaction mixture was spotted onto a 1 cm² piece of Whatman 3MM filterpaper. The filter paper was immediately dipped in 10% ice-cold TCA, andthen kept on ice in a plastic tray. After all reactions w ere run, thefilter papers were washed at 4 C with stirring in a large excess of 10%TCA, similarly washed twice at 4 C with 100% methanol, and then vacuumdried at 45° C. for 1 hr to remove the ³H-methanol produced by PP2Ademethylation. The dried filter papers then were assayed forradioactivity by submerging them in scintillation fluid and performingscintillation counting. This procedure is easily adapted to a 96-wellformat to allow high-throughput screening (described below).

As used herein to refer to assay results, the term “inhibition activity”refers to the following relationship:${{Inhibition}\quad{activity}} = {1 - \frac{\begin{pmatrix}{{amount}\quad{of}\quad{methyl}\text{-}} \\{{PP}\quad 2A\quad{demethylated}\quad{with}\quad{inhibitor}}\end{pmatrix}}{\begin{pmatrix}{{amount}\quad{of}\quad{methyl}\text{-}} \\{{PP}\quad 2A\quad{demethylated}\quad{without}\quad{inhibitor}}\end{pmatrix}}}$

The activity of an inhibitor according to the present invention isdefined herein as 1 unit of inhibitor inhibits 50% of the demethylationin 10 μl of reaction mixture in 10 minutes.

Drip-brewed coffee (prepared from Sumatra coffee purchased fromStarbucks Coffee) was tested in an initial screen. The filter paperassay described above was used to assay 1 μl of Sumatra coffee per 10 μlof assay mixture. The assay showed that Sumatra coffee contained aninhibition activity of 0.83 (i.e., 83% inhibition relative to control).

Based on this significant inhibition of PPME, we tested an ethanolextract of Sumatra coffee that showed an even more significantinhibitory effect. 10 g of ground Sumatra coffee bean (Starbucks) wasextracted in ethanol at 80° C. for 5 minutes with stirring. Aftercooling, the extract was filtered through a Whatman No. 54 paper filter.The filtered extract was rotary evaporated at 40° C. until the volumewas reduced to approximately 1 ml. The liquid phase was recovered anddiluted into 50 mM MOPS buffer, 1 mM EDTA, pH 7.2.

FIG. 3 shows the results of an assay using the filter paper method toassay 1 μl of extract per 10 μl of assay mixture. The results show thatthis ethanol extract of Sumatra coffee grounds shows aconcentration-dependent inhibition activity. The inhibitor effect seenis due to a small molecule that is soluble in organic solvents and isnot caffeine. The Concentration of pure caffeine required for 50%inhibition of PPME was >440 μM. Inhibitory activity was present atsimilar levels in both regular and decaffeinated coffee.

Example 5 Assay for Methyltransferase Activity

This example provides guidance for determining activation of PPMT.Purified PP2A AC dimers (see Example 3) are mixed with the purified PPMT(prepared as described in Example 1) and ³H-methyl-labeled S-adenosylmethionine and incubated at 37 C. After several minutes aliquots wereremoved, protein was precipitated with 10% trichloroacetic acid (TCA),washed with 10% TCA, then washed with 100% methanol, and ³H-methyl-PP2Awas assayed by liquid scintillation counting (Tolstykh, T. et al., EMBOJ. 19 (21): 5682-91 (2000).

Example 6 Purification of the Component(s) in Coffee that Inhibit PP2AMEase

This example provides methods by which inhibition activity can bemeasured for a larger number of compounds in parallel in a 96-well plateformat, and provides methods through which the inhibitory component(s)in coffee may be selectively enriched or purified.

(a) Inhibitory Effect of Coffee Component(s) on PPME Assayed in a96-Well Format:

The inhibitory effect of coffee component(s) on PPME was measured usingthe filter paper method as adapted to a 96-well plate format. Equimolaramounts of PP2A and [³H]-SAM and a two-fold molar excess of PPMT wereincubated at 37° C. for 30 min., which is the time when the methylationreaction reaches its equilibrium, and PP2A is close to 100% methylated.Methylated PP2A was separated from PPMT by diluting the reaction mixturein buffer with Ni-NTA resin and bovine serum albumin (BSA) and shakingat room temperature for 30 min. The supernatant containing methylatedPP2A was collected, and Ni-resin to which PPMT bound was discarded.

96-well Millipore filter plates were used to assay demethylation ofmethylated PP2A. Methylated PP2A was mixed with PPME at 37° C. Atreaction times of 5 and 10 min, 10 μl of reaction mixture was spottedinto a well of the filter plate containing cold 25% TCA to stop thedemethylating reaction. The filter in each well was washed with 5% TCA,then with 70% ethyl alcohol, and finally air-dried. Scintillation fluidwas added into the wells, and the radioactivity due to bound³H-methyl-PP2A was counted using a Packard TopCount scintillationcounter.

(b) Purification of Coffee Components Inhibitory to PPME Using EthylAcetate Partitioning.

The inhibition activity in coffee may be purified from extracts ofeither whole coffee or instant coffee by a selective partitioning intoethyl acetate at low versus high pH.

10 g of ground Sumatra coffee bean (Starbucks) was extracted withethanol at 80 C for 5 minute with stirring. After cooling, the extractwas filtered through a Whatman No. 54 paper filter. The filtered extractwas rotary evaporated at 40 C until the volume was reduced toapproximately 1 ml. 200 μl of this concentrated extract was mixed withan equal volume of 50 mM MOPS buffer, pH 7.2 with 1 mM EDTA (Buffer A),0.1M HCl, or 0.1M NaOH, and then extracted with 1 ml of ethyl acetate.After separation by centrifugation, the aqueous and organic phases ineach tube were collected, dried under vacuum in a Speedvac concentrator,resuspended in Buffer A to a volume of 2 ml, and the pH adjusted to 7.2.This extract was assayed using the filter paper method with a volume of1 μl of extract per 10 μl of sample volume. The results of this assaydemonstrated an inhibition activity of about 1 (100% inhibition) for theorganic phase in the presence of Buffer A or HCl, an inhibition activityof about 0.2 for the organic phase in the presence of NaOH, aninhibition activity of about 0 from the aqueous phase in the presence ofBuffer A or HCl, and an inhibition activity of about 1 from the aqueousphase in the presence of NaOH.

1 g of decaffeinated instant coffee also was dissolved in a 10 ml finalvolume of 0.1M HCl (“acidic coffee solution”). The final pH of theacidic coffee solution was 3.0, and an amount of insoluble material wasremoved by centrifugation. The amount of insoluble material was largerthan the amount of insoluble material present after dissolving 1 g ofinstant coffee in 10 ml of hot deionized water at pH 5 (the“water-extracted coffee solution”). 5 ml of the acidic coffee solutionwas extracted with 4 ml of ethyl acetate, and the organic phaserecovered. The organic phase then was extracted with an equal volume of0.1M NaOH. The final extract was clearer and much lighter in color thanwas instant coffee dissolved in water or HCl. These extracts wereassayed using the 96 well plate method and a diluted extract equivalentto a volume of about 0.1 μl of extract per 10 μl of assay mixture. Underthese conditions, the water extracted coffee solution had an inhibitionactivity of about 0.78, the acidic coffee solution had an inhibitionactivity of 0.44, and the NaOH extract of the organic phase had aninhibition activity of 0.47. These results and the results describedabove demonstrate that the inhibitory compounds from coffee can bepurified by selective partitioning into ethyl acetate at acidic pH andselective partitioning into the aqueous phase at basic pH.

(c) Purification of Coffee Components Inhibitory to PPME Using EthanolExtraction, Water Wash and Reverse-Phase Separation.

10 g of decaffeinated instant coffee (Taster's Choice) was boiled (80°C.) three times in 100 ml 100% ethanol for 5 min. Each ethanol extractwas centrifuged at 3,000 rpm, 4° C. for 30 min, the supernatantcollected, and the coffee-solids-containing pellet used for thesubsequent extraction. Inhibition activity present in these extracts wasmeasured by the 96 well plate assay described in (a) above.

FIG. 4A shows the presence of a methylation modifying compoundpossessing PP2A methylesterase inhibition activity in an extractprepared from instant coffee by ethanol extraction. FIG. 4A shows PPMEinhibition activity in each crude ethanol extract after three 100 mlethanol extractions, 300 ml total volume. The extracted inhibitionactivities in the second and third extracts were less than in the firstextract, such that activity present in the second and third extractionswas about half of the previous extract's activity. Based on thisobservation, 200 ml EtOH should be enough to extract the majority of themethylation modifying activity in 10 g of instant coffee.

Instant coffee was boiled (80° C.) once in 100 ml ethanol for 5 minutesand twice time in 50 ml ethanol for 5 min each time. Each extract wascentrifuged at 3,000 rpm, 4° C. for 30 min, and the supernatantscombined to yield a crude extract with an approximate volume of 200 ml.The inhibition activity in the final extract was measured by the 96 wellplate assay described above.

FIG. 4B shows PP2A methyl esterase inhibition activity in crude ethanolextract after these extractions. As shown in FIG. 4B, about 10⁶ unitstotal inhibitory units were extracted in 200 ml EtOH from 10 g coffee.

The ethanol was filtered through Fisher Scientific medium porosity typeP5 filter paper (P5 filter paper), rotary-evaporated and the brown,thick residue collected. The residue weighed 1.18 g. The dried coffeeresidue obtained from the crude ethanol extract was washed four timeswith 50 ml of doubly deionized water with pH <5. The water solutionswere filtered through P5 filter paper. 50 ml of ethanol was used todissolve components that stuck to the flask, and another 20 ml ethanolwas used to wash the filter paper. The material dissolved in ethanol andnot dissolvable in water was much lighter in color than was the crudeextract.

The PP2A demethylation inhibition activity of the water washes, ethanolused to wash the flask, and ethanol used to wash the filter paper areshown in FIG. 4C. Only about 10% of the total inhibition activity in thecrude ethanol extract was lost in the water-wash step. The residuedissolved from the flask and filter paper with ethanol was rotaryevaporated to yield a final coffee extract containing nearly all theinhibitory activity and having a mass of about 0.1 g.

A solid-phase extraction cartridge containing C-18 resin (Waters)equilibrated with 50% methanol plus 0.1% trifluoroacetic acid (TFA) wasused to further purify the inhibitory compound in the final coffeeextract. The ethanol in the final coffee extract was rotary-evaporated.The residue was dissolved in 50% methanol plus 0.1% TFA and loaded onthe C-18 cartridge. The cartridge then was washed with three columnvolumes of 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90%methanol, 100% methanol, and 100% acetonitrile, all with 0.1% TFA.Materials eluted with each mobile phase were collected as separatefractions. Water and organic solvents in each fraction were rotaryevaporated, and the residue was dissolved in ethanol.

FIG. 4D shows the elution profile of inhibitory activity. Eluentcorresponding to 0.5 μl of the final coffee extract was used per 10 μlof PP2A demethylation reaction. The 90% methanol and 100% methanolfractions contained most of the PPME inhibition activity present in thefinal coffee extract and little or no activity remained to be eluted bythe acetonitrile.

Example 7 Identification of PP2A Inhibitory Activity in Botanicals

We have used the assay described above in Example 4 to screencommercially available herbal substances for the presence of a PP2Ademethylation inhibitor compound. Each of these substances has beenreported to have properties that affect the health and well-being ofhuman subjects. For example,

Allicin is an antibiotic and antifungal compound obtained from garlic.

Artichoke powder is believed to have hepatoprotective, antioxidant, andhypocholesterolemic properties.

Bacopa monniera is an herb used in India for memory, epilepsy, and as amild sedative. Bacopa commonly grows in marshy areas throughout India.Bacopa is believed to have strong antioxidant properties, protect mentalfunction and improve learning skills.

Butcher's broom, an herbal extract that typically contains 10% saponinglycosides as active components, has been reported to haveanti-inflammatory properties.

Cocoa powder cocoa contains a high content of phenolic phytochemicals,or flavonoids, indicating the presence of known antioxidants that mayprotect against cancer, heart disease and other ailments.

Flaxseed oil, which is obtained by extracting the oil from flaxseeds isbelieved to stimulate the immune system and to have antioxidant,cholesterol and triglyceride lowering and anti-tumor properties.

Grape seed extract contains oligomeric proanthocyanidins (OPCs), a classof flavonoid complexes that act as antioxidants in the body. It isbelieved that OPCs may help protect against the effects of internal andenvironmental stresses and may counter the negative effects of highcholesterol on the heart and blood vessels.

Purportedly, nutritional supplementation of inositol may affect behaviorand may have anti-depressant and anti-anxiety activities.

St. John's Wort, Hypericum perforatum, is an herbal product sold as anover-the-counter treatment for depression. It is being studied for itsability to lessen certain side effects of cancer treatment.

The antioxidants in green tea, black tea and red tea purportedly havesignificant health benefits, including the ability to prevent cancer andheart disease.

Tobacco extract has been shown to produce a biphasic effect onmacrophage respiration: a stimulation at low concentrations and aninhibition at higher concentrations.

The putative health affects of ginseng, gingko, huperzine, Vitamin E(DL-alpha-tocopherol), and choline already have been described above.

Extracts typically were made from the contents of one tablet, capsule orcaplet by heating in 1 ml of pure (200 proof) ethanol for 30 minutes at80° C. before centrifugation for 5 to 10 minutes at 12,000 rpm in aSorvall MC-12V centrifuge to remove insoluble material. In the tablebelow, this is indicated as a “standard ethanol” extraction. Extractsfor tea were made by heating in 10 ml of doubly deionized water at 80°C. for 30 minutes before centrifugation. This is denoted in the table asa “standard water” extraction. Other extractions are as specified, andpure or nearly pure compounds were dissolved in the solvent listed. Aportion of each extract was dried to determine the mass of dissolvedmaterial in the extract. TABLE 1 Commercially available herbalsubstances screened for the presence of a PP2A demethylation inhibitorcompound. Sample Code Substance Extraction method Source #3 GingkoBiloba leaf extract: 60 mg Standard ethanol Sundown per tablet. 24%flavonol glycosides (14.4 mg), 6% terpene lactones (3.6 mg). Alsocontains 14 support ingredients. #4 Allicin, (garlic bulb extract): 130mg/ Standard ethanol Zhang capsule. Also contains cellulose. #5Huperzine A, Gotu Kola Dissolved in ethanol Solaray (Centella asiatica,aerial part) 285 mg, Huperzine 50 μg, lecithin 100 mg/ capsule. Alsocontains 3 support ingredients. #6 Bacopa, Himalaya (Bacopa Standardethanol Brahmi moniera fennel leaf extract): 500 mg/ capsule. #7Butcher's Broom: root extract Standard ethanol TWINLAB with otheringredients, no amount Nature's Herbs was shown on the bottle label.Also it is not clear whether or not support ingredient was included. #10Flaxseed oil - contains 1425 mg Dissolved in DMSO Solgar of linolenicacid, 450 mg of oleic acid, 400 mg of linoleic acid, 150 mg of palmiticacid, 75 mg of stearic acid/2 softgels. Other ingredients: flaxseed oil.Oil volume is about 1.4 ml/soft gel. #12-1 DL-α-tocopherol,, 95%, MWDissolved in ethanol Sigma (T3251- 431, prepared from synthetic 25G)phytol. #21 Ginseng - 100 mg of Ginseng Standard ethanol Your Lifeextract/caplet, 7% ginsenosides in the extract, also contains calciumcarbonate et al 16 ingredients #22 Grape Seed Extract, 100 mg ofStandard ethanol Wegmans grape seed extract (85% polyphenols)/tablet,also contains calcium carbonate et al 11 ingredients. #23 St. John'sWort, 300 mg of St. Standard ethanol Wegman's John's wort extract (0.3%total dianthrones)/caplet,, also contains cellulose et al 11ingredients. #24 Artichoke-powder, 100 mg of Standard ethanol Nature'sHerbs artichoke (aerial part) extract and 375 mg of artichoke (aerialpart)/ capsule, also contains vitamin E et al 4 ingredients. #25 Choline& Inositol Capsules, Extracted with room Twinlab choline 250 mg andinostol 250 mg/ temperature ethanol; capsule, also contains gelatinotherwise standard. et al 5 ingredients. #26 Green Tea, 1.5 g/bag,ingredients: Standard water Wissotzky green tea, verbena, lemongrass,lemon flavor. #27 Red Bush Tea, 2 g/bag, Standard water BrassicaIngredients: South African rooibos (Aspalathus linearis) and lemongrasswith 15 mg of SGS made from broccoli seed. (no caffeine) #28 Black Tea,2.1 g/bag, Chinese Standard water Brassica black tea with 15 mg of SGSmade from broccoli seed, caffeine #30E Tobacco No additive. Sliced 820mg extracted with 10 ml Natural tobacco leaves. ethanol for 60 min atAmerican Spirit 80° C. #31 Cocoa, unsweetened -. 150 mg of the powderwas Ghiradelli extracted using standard Chocolate Co. ethanolextraction. CCM Curcumin Dissolved in ethanol. LKT Laboratories, Inc.CGA Chlorogenic acid (“CGA”), Dissolved in 50% ethanol. Acros Organicspredominantly trans(ex coffee seeds) 99%, MW = 354.3 DHCA3,4-Dihdroxycinnamic acid Dissolved in 50% ethanol. Acros Organics(“DHCA”), predominantly trans isomer 99+%, MW = 180.6 HMCA4-Hydroxy-3-methoxycinnamic Dissolved in 50% ethanol. Acros Organicsacid (“HMCA”), 99%, MW = 194 QA D(−)-Quinic acid (“QA”), 98%, Dissolvedin buffer. Acros Organics MW192.17,

The concentration of each substance that resulted in an inhibitionactivity of 0.5 (50% inhibition of PPME) was determined using the filterpaper method described above in Example 4, and is hereafter referred toas the IC₅₀. The results, which are expressed as dry extract weight/mlexcept as noted, are shown in Table 2. TABLE 2 Effect of Substances onPPME activity (assayed against PPME at 85 nM by the filter paper method.Sample Code Substance name IC₅₀ #3 Gingko (extract) 106 μg/ml #4 Allicin(extract) 100 μg/ml #5-1 Huperzine No inhibition at 40 μM #6 Bacopa(extract) 63 μg/ml #7 Butcher's broom 37 μg/ml #10 Flaxseed oil 1.3mg/ml #12-1 DL-α-tocopherol 35 μM #21 Ginseng (extract) 360 μg/ml #22Grape seed (extract) 25 μg/ml #23 St. John's wort (extract) 22 μg/ml #24Artichoke-powder (extract) 260 μg/ml #25 Choline & inositol caps(extract) 75 μg/ml #26 Green tea (extract) 179 μg/ml #27 Red bush tea(extract) 45 μg/ml #28 Black tea (extract) 50 μg/ml #30E Tobacco(extract) 344 μg/ml #31 Cocoa, Ghirardelli Chocolate Co., 92 μg/mlunsweetened CCM-L Curcumin-(LKT Co.) 771 μM CGA Chlorogenic acid >1 mMDHCA Dihydroxycinnamic acid >1 mM HMCA Hydromethoxycinnamic acid >1 mMQA Quinic acid >10 mM

For purposes of this table, the unit of concentration in the above table(μg/ml or mg/ml) refers to dried extract weight (DEW) unless otherwisenoted. A “>” symbol before the IC₅₀ value means that the IC₅₀concentration is higher than the value tested. Most of these IC₅₀ valuesare high compared to the concentration of the AC dimer of PP2A (30-50nM) in the reaction mixture.

From these results we conclude that all of these compounds excepthuperzine, chlorogenic acid, dihydroxycinnamic acid, HMCA, and quinicacid contain significant PPME inhibitory activity. Since the measuredactivity is dependent on the extraction procedure, the assay describedcan be used to optimize the extraction protocol and to select varietiesor sources of these compounds that contain the most concentrated PPMEinhibitory activity.

The present invention has been described with reference to the specificembodiments thereof, it should be understood by those skilled in the artthat various changes may be made and equivalents may be substitutedwithout departing from the true spirit and scope of the Invention. Inaddition, many modifications may be made to adapt a particularsituation, material, composition of matter, process, process step orsteps, to the objective, spirit and scope of the present invention. Allsuch modifications are intended to be within the scope of the followingclaims.

1. A composition for promoting general health and well-being in amammalian subject, including a human, comprising an isolated methylationmodifying compound.
 2. The composition according to claim 1, wherein thecompound is isolated from a botanical raw material.
 3. The compositionaccording to claim 1, wherein the compound is isolated from a microbialraw material.
 4. The composition according to claim 1, wherein thecomposition inhibits a demethylating activity of a demethylating enzymethat acts on a protein phosphatase 2A enzyme and thereby stimulatesmethylation of the protein phosphatase 2A enzyme.
 5. The compositionaccording to claim 4, wherein the composition inhibits at least about50% of the demethylating activity of the demethylating enzyme.
 6. Thecomposition according to claim 4, wherein the demethylating enzyme is aprotein phosphatase 2A specific protein methylesterase.
 7. Thecomposition according to claim 6, wherein the demethylating activity ofthe protein phosphatase 2A specific protein methylesterase is determinedby measuring levels of protein phosphatase 2A methyl esterification. 8.The composition according to claim 2, wherein the botanical raw materialcomprises at least one substance selected from the group consisting ofgingko, allicin, bacopa; butcher's broom; flaxseed oil; a tocopherol;vitamin E; ginseng, grape seed, St. John's wort; artichoke-powder;choline; inositol; coffee; tea; tobacco; and cocoa.
 9. The compositionaccording to claim 2, wherein the botanical raw material comprises afruit of a species of plant genus Coffea.
 10. The composition accordingto claim 9, wherein the fruit of the plant genus Coffea is a coffeebean.
 11. The composition according to claim 2, wherein the botanicalraw material comprises a nutritional supplement.
 12. The compositionaccording to claim 11, wherein the nutritional supplement comprises atleast one tocopherol.
 13. The composition according to claim 11, whereinthe nutritional supplement comprises Vitamin E.
 14. The compositionaccording to claim 2, wherein the botanical raw material comprises aleaf of a tobacco plant.
 15. The composition according to claim 2,wherein the botanical raw material comprises a ground coffee.
 16. Thecomposition according to claim 2, wherein the botanical raw materialcomprises an instant coffee.
 17. The composition according to claim 2,wherein the methylation modifying compound is isolated from thebotanical raw material by extracting the botanical raw material with apolar solvent.
 18. The composition according to claim 17, wherein thepolar solvent is a lower alcohol.
 19. The composition according to claim18, wherein the lower alcohol is ethanol.
 20. The composition accordingto claim 17, wherein the polar solvent is ethyl acetate.
 21. Thecomposition according to claim 20, wherein the methylation modifyingcompound is soluble in ethyl acetate below a pH of about 5 and insolublein ethyl acetate above a pH of about
 10. 22. The composition accordingto claim 1, wherein the composition is essentially free of at least onesubstance selected from the group consisting of caffeine, caffeic acidand chlorogenic acid.
 23. A comestible for promoting general health andwell-being in a mammalian subject, including a human, comprising acomposition containing an isolated methylation modifying compound. 24.The comestible according to claim 23, wherein the compound is isolatedfrom a botanical raw material.
 25. The comestible according to claim 23,wherein the compound is isolated from a microbial raw material.
 26. Thecomestible according to claim 23, wherein the composition inhibits ademethylating activity of a demethylating enzyme that acts on a proteinphosphatase 2A enzyme and thereby stimulates methylation of the proteinphosphatase 2A enzyme.
 27. The comestible according to claim 26, whereinthe composition inhibits at least about 50% of the demethylatingactivity of the demethylating enzyme.
 28. The comestible according toclaim 26, wherein the demethylating enzyme is protein phosphatase 2Aspecific protein methylesterase.
 29. The comestible according to claim26, wherein the level of protein methylesterase demethylating activityis determined by measuring levels of protein phosphatase 2A methylesterification.
 30. The comestible according to claim 24, wherein thebotanical raw material comprises at least one substance selected fromthe group consisting of gingko, allicin, bacopa; butcher's broom;flaxseed oil; a tocopherol; vitamin E; ginseng, grape seed, St. John'swort; artichoke-powder; choline; inositol; coffee; tea; tobacco; andcocoa.
 31. The comestible according to claim 24, wherein the botanicalraw material comprises a fruit of a species of plant genus Coffea. 32.The comestible according to claim 31, wherein the fruit of the plantgenus Coffea is a coffee bean.
 33. The comestible according to claim 24,wherein the botanical raw material comprises a nutritional supplement.34. The comestible according to claim 33, wherein the nutritionalsupplement comprises at least one tocopherol.
 35. The comestibleaccording to claim 33, wherein the nutritional supplement comprisesVitamin E.
 36. The comestible according to claim 24, wherein thebotanical raw material comprises a leaf of a tobacco plant.
 37. Thecomestible according to claim 24, wherein the botanical raw materialcomprises a ground coffee.
 38. The comestible according to claim 24,wherein the botanical raw material comprises an instant coffee.
 39. Thecomestible according to claim 24, wherein the methylation modifyingcompound is isolated from the botanical raw material by extracting thebotanical raw material with a polar solvent.
 40. The comestibleaccording to claim 39, wherein the polar solvent is a lower alcohol. 41.The comestible according to claim 40, wherein the lower alcohol isethanol.
 42. The comestible according to claim 39, wherein the polarsolvent is ethyl acetate.
 43. The comestible according to claim 42,wherein the methylation modifying compound is soluble in ethyl acetatebelow a pH of about 5 and insoluble in ethyl acetate above a pH of about10.
 44. The comestible according to claim 23, wherein the composition isessentially free of at least one substance selected from the groupconsisting of caffeine, caffeic acid and chlorogenic acid.
 45. Thecomestible according to claim 23, wherein the comestible is a beverage.46. The comestible according to claim 45, wherein the beverage isselected from the group consisting of a drink comprising water, a fruitdrink, a coffee, a tea, an energy drink, a baby formula, an adultnutritional drink, a health drink, and a sports drink.
 47. Thecomestible according to claim 23, wherein the comestible is a food. 48.The comestible according to claim 47, wherein the comestible is acereal.
 49. The comestible according to claim 23, wherein the comestibleis a chewing gum.
 50. The comestible according to claim 23, wherein thecomestible is a candy.
 51. The comestible according to claim 23, whereinthe comestible is an ingredient of an inhalant.
 52. The comestibleaccording to claim 23, wherein the comestible is an ingredient of atransdermal delivery system.
 53. A pharmaceutical preparation forpromoting general health and well-being in a mammalian subject,including a human, comprising a cognitive function-enhancing amount of acomposition containing an isolated methylation modifying compound and apharmaceutically acceptable carrier.
 54. The pharmaceutical preparationaccording to claim 53, wherein the compound is isolated from a botanicalraw material.
 55. The pharmaceutical preparation according to claim 53,wherein the compound is isolated from microbial raw material.
 56. Thepharmaceutical preparation according to claim 53, wherein the compoundinhibits a demethylating enzyme that acts on a protein phosphatase 2Aand thereby stimulates methylation of the protein phosphatase 2A enzyme.57. The pharmaceutical preparation according to claim 56, wherein thecomposition inhibits at least about 50% of the demethylating activity ofthe demethylating enzyme.
 58. The pharmaceutical preparation accordingto claim 56, wherein the demethylating enzyme is a protein phosphatase2A specific protein methylesterase.
 59. The pharmaceutical preparationaccording to claim 58, wherein the demethylating activity of the proteinphosphatase 2A specific protein methylesterase is determined bymeasuring levels of protein phosphatase 2A methyl esterification. 60.The composition according to claim 54, wherein the botanical rawmaterial comprises at least one substance selected from the groupconsisting of gingko; allicin, bacopa; butcher's broom; flaxseed oil; atocopherol; vitamin E; ginseng, grape seed, St. John's wort;artichoke-powder; choline, inositol; tea; tobacco; and cocoa.
 61. Thepharmaceutical preparation according to claim 54, wherein the botanicalraw material comprises a fruit of a species of plant genus Coffea. 62.The pharmaceutical preparation according to claim 61, wherein the fruitof the plant genus Coffea is a coffee bean.
 63. The pharmaceuticalpreparation according to claim 54, wherein the botanical raw materialcomprises a ground coffee.
 64. The pharmaceutical preparation accordingto claim 54, wherein the botanical raw material comprises an instantcoffee.
 65. The pharmaceutical preparation according to claim 54,wherein the botanical raw material is a nutritional supplement.
 66. Thepharmaceutical preparation according to claim 65, wherein thenutritional supplement comprises at least one tocopherol.
 67. Thepharmaceutical preparation according to claim 65, wherein thenutritional supplement comprises vitamin E.
 68. The pharmaceuticalpreparation according to claim 54, wherein the botanical raw materialcomprises a leaf of a tobacco plant.
 69. The pharmaceutical preparationaccording to claim 54, wherein the methylation modifying compound isisolated from the botanical raw material by extracting the botanical rawmaterial with a polar solvent.
 70. The pharmaceutical preparationaccording to claim 69, wherein the polar solvent is a lower alcohol. 71.The pharmaceutical preparation according to claim 70, wherein the loweralcohol is ethanol.
 72. The pharmaceutical preparation according toclaim 69, wherein the polar solvent is ethyl acetate.
 73. Thepharmaceutical preparation according to claim 72, wherein themethylation modifying compound is soluble in ethyl acetate below a pH ofabout 5 and insoluble in ethyl acetate above a pH of about
 10. 74. Thepharmaceutical preparation according to claim 53, wherein thepreparation is essentially free of at least one substance selected fromthe group consisting of caffeine, caffeic acid and chlorogenic acid. 75.The pharmaceutical preparation according to claim 53, wherein thepreparation is administered orally.
 76. The pharmaceutical preparationaccording to claim 53, wherein the preparation is in the form of apowder.
 76. The pharmaceutical preparation according to claim 53,wherein the preparation is in the form of a tablet.
 77. Thepharmaceutical preparation according to claim 53, wherein thepreparation is in the form of a capsule.
 78. The pharmaceuticalpreparation according to claim 53, wherein the preparation is in theform of a lozenge.
 79. The pharmaceutical preparation according to claim53, wherein the preparation is in the form of a suppository.
 80. Amethod for promoting general health and well-being in a mammaliansubject, including a human, the method comprising the steps (a)isolating a methylation modifying compound; and (b) administering acognitive function-enhancing amount of a composition comprising theisolated methylation modifying compound to a subject in need thereof.81. The method according to claim 80, wherein in step (a) themethylation modifying compound is isolated from a botanical rawmaterial.
 82. The method according to claim 80, wherein in step (a) themethylation modifying compound is isolated from a microbial rawmaterial.
 83. The method according to claim 80, wherein the methylationmodifying compound inhibits a demethylating enzyme that acts on aprotein phosphatase 2A enzyme and thereby stimulates methylation of theprotein phosphatase 2A enzyme.
 84. The method according to claim 83,wherein the composition inhibits at least about 50% of the demethylatingactivity of the demethylating enzyme.
 85. The method according to claim83, wherein the demethylating enzyme is a protein phosphatase 2Aspecific protein methylesterase.
 86. The method according to claim 85,further comprising the step of (c) determining the demethylatingactivity of the protein phosphatase 2A specific protein methylesterase.87. The method according to claim 80, wherein the cognitionfunction-enhancing amount of the composition is from about 1 mg to about10 g.
 88. The method according to claim 81, wherein the botanical rawmaterial comprises at least one substance selected from the groupconsisting of gingko, allicin, bacopa; butcher's broom; flaxseed oil; atocopherol; vitamin E; ginseng, grape seed, St. John's wort;artichoke-powder; choline; inositol; tea; tobacco; and cocoa.
 89. Themethod according to claim 81, wherein the botanical raw materialcomprises a fruit of a species of plant genus Coffea.
 90. The methodaccording to claim 89, wherein the fruit of the plant genus Coffea is acoffee bean.
 91. The method according to claim 81, wherein the botanicalraw material comprises a ground coffee.
 92. The method according toclaim 81, wherein the botanical raw material comprises an instantcoffee.
 93. The method according to claim 81, wherein the botanical rawmaterial comprises a nutritional supplement.
 94. The method according toclaim 93, wherein the nutritional supplement comprises at least onetocopherol.
 95. The method according to claim 93, wherein thenutritional supplement comprises vitamin E.
 96. The method according toclaim 81, wherein the botanical raw material comprises a leaf of atobacco plant.
 97. The method according to claim 81, step (a) furthercomprising the step of extracting the botanical raw material with apolar solvent.
 98. The method according to claim 97, wherein the polarsolvent is a lower alcohol.
 99. The method according to claim 98,wherein the lower alcohol is ethanol.
 100. The method according to claim97, wherein the polar solvent is ethyl acetate.
 101. The methodaccording to claim 100, step (a) further comprising the steps ofsolubilizing the methylation modifying compound in ethyl acetate below apH of about 5 and partitioning the methylation modifying compoundbetween an aqueous phase and ethyl acetate above a pH of about
 10. 102.The method according to claim 80, wherein the extracted composition isessentially free of at least one substance selected from the groupconsisting of caffeine, caffeic acid and chlorogenic acid.
 103. Themethod according to claim 80, wherein in step (b), the composition isadministered orally.
 104. The method according to claim 80, wherein thecomposition is a powder.
 105. The method according to claim 80, whereinthe composition is in the form of a tablet
 106. The method according toclaim 80, wherein the composition is in the form of a capsule.
 107. Themethod according to claim 80, wherein the composition is in the form ofa lozenge.
 108. The method according to claim 80, wherein thecomposition is in the form of a suppository.
 109. A composition forpromoting general health and well-being in a mammalian subject,including a human, prepared by a process, the process comprising thesteps of: a) contacting a botanical raw material with a polar solventfor a time sufficient to form an extract solution from the botanical rawmaterial; b) isolating the extract solution and removing the polarorganic solvent to form a concentrated primary extract; and c)dissolving the composition in a solvent at a pH ranging from about 7 toabout 10 to form a concentrated purified extract.
 110. The compositionaccording to claim 109 wherein the polar solvent in step (a) of theprocess is a lower alcohol.
 111. The composition according to claim 110,wherein the lower alcohol is ethanol.
 112. The composition according toclaim 119, wherein the polar solvent in step (a) of the process is ethylacetate.
 113. The composition according to claim 112, wherein step (a)of the process further comprises the step of solubilizing thecomposition in ethyl acetate below a pH of about
 5. 114. The compositionaccording to claim 112, wherein step (b) of the process furthercomprises the step of partitioning the methylation modifying compoundbetween an aqueous phase and ethyl acetate above a pH of about
 10. 115.The composition according to claim 109, wherein the composition inhibitsa demethylating activity of a demethylating enzyme that acts on aprotein phosphatase 2A enzyme and thereby stimulates methylation of theprotein phosphatase 2A enzyme.
 116. The composition according to claim115, wherein the composition inhibits at least about 50% of thedemethylating activity of the demethylating enzyme.
 117. The compositionaccording to claim 115, wherein the demethylating enzyme is a proteinphosphatase 2A specific protein methylesterase.
 118. The compositionaccording to claim 109, wherein the botanical raw material comprises atleast one substance selected from the group consisting of gingko;allicin; bacopa; butcher's broom; flaxseed oil; a tocopherol; vitamin E;ginseng; grape seed; St. John's wort; artichoke-powder; choline;inositol; coffee; tea; tobacco; and cocoa.
 119. The compositionaccording to claim 109, wherein the botanical raw material comprises afruit of a species of plant genus Coffea.
 120. The composition accordingto claim 119, wherein the fruit of the plant genus Coffea is a coffeebean.
 121. The composition according to claim 109, wherein the botanicalraw material comprises a ground coffee.
 122. The composition accordingto claim 109, wherein the botanical raw material comprises an instantcoffee.
 123. The composition according to claim 109, wherein thebotanical raw material comprises a nutritional supplement.
 124. Thecomposition according to claim 123, wherein the nutritional supplementcomprises at least one tocopherol.
 125. The composition according toclaim 123, wherein the nutritional supplement comprises Vitamin E. 126.The composition according to claim 109, wherein the botanical rawmaterial comprises a leaf of a tobacco plant.
 127. The compositionaccording to claim 109, wherein the concentrated purified extract isessentially free of at least one substance selected from the groupconsisting of caffeine, caffeic acid and chlorogenic acid.
 128. Thecomposition according to claim 109, wherein the composition isadministered orally.
 129. The composition according to claim 109,wherein the composition is in the form of a powder.
 130. The compositionaccording to claim 109, wherein the composition is in the form of atablet.
 131. The composition according to claim 109, wherein thecomposition is in the form of a capsule.
 132. The composition accordingto claim 109, wherein the composition is in the form of a lozenge. 133.The composition according to claim 109, wherein the composition is inthe form of a suppository.
 134. A method of treating or preventingcognitive effects of a disease, condition, or disorder involvingdefective methylation metabolism in a mammalian subject, including ahuman, the method comprising the steps of (a) isolating a methylationmodifying compound; and (b) administering a therapeutically effectiveamount of a composition comprising the isolated methylation modifyingcompound to a subject in need thereof.
 135. The method according toclaim 134, wherein in step (a) the methylation modifying compound isisolated from a botanical raw material.
 136. The method according toclaim 134, wherein in step (a) the methylation modifying compound isisolated from a microbial raw material.
 137. The method according toclaim 134, wherein the disease, condition, or disorder is associatedwith higher than normal levels of plasma homocysteine.
 138. The methodaccording to claim 134, wherein the disease condition or disorder is atleast one disease, condition or disorder selected from the groupconsisting of a cardiac disorder; atherosclerosis; a neurodegenerativedisorder; a cerebrovascular disorder; a neuropsychiatric disorder; anddiabetes.
 139. The method according to claim 134, wherein themethylation modifying compound inhibits a demethylating activity of ademethylating enzyme that acts on a protein phosphatase 2A enzyme andthereby stimulates methylation of the protein phosphatase 2A enzyme.140. The method according to claim 139, wherein the composition inhibitsat least about 50% of the demethylating activity of the demethylatingenzyme.
 141. The method according to claim 139, wherein thedemethylating enzyme is a protein phosphatase 2A specific proteinmethylesterase.
 142. The method according to claim 134, wherein thetherapeutically effective amount of the composition is from about 1 mgto about 10 g of the composition.
 143. The method according to claim134, wherein the botanical raw material comprises at least one substanceselected from the group consisting of gingko, allicin, bacopa; butcher'sbroom; flaxseed oil; a tacopherol; Vitamin E; ginseng, grape seed, St.John's wort; artichoke-powder; choline; inositol; tea; tobacco; andcocoa.
 144. The method according to claim 134, wherein the botanical rawmaterial comprises a fruit of a species of plant genus Coffea.
 145. Themethod according to claim 144, wherein the fruit of the plant genusCoffea is a coffee bean.
 146. The method according to claim 134, whereinthe botanical raw material comprises a ground coffee.
 147. The methodaccording to claim 134, wherein the botanical raw material comprises aninstant coffee.
 148. The method according to claim 134, wherein thebotanical raw material comprises a nutritional supplement.
 149. Themethod according to claim 148, wherein the nutritional supplementcomprises at least one tocopherol.
 150. The method according to claim148, wherein the nutritional supplement comprises Vitamin E.
 151. Themethod according to claim 134, wherein the botanical raw materialcomprises a leaf of a tobacco plant.
 152. The method according to claim134, step (a) further comprising the step of extracting the botanicalraw material with a polar solvent.
 153. The method according to claim152, wherein the polar solvent is a lower alcohol.
 154. The methodaccording to claim 152, wherein the polar solvent is ethyl acetate. 155.The method according to claim 154, wherein step (a) further comprisesthe steps of solubilizing the methylation modifying compound in ethylacetate below a pH of about 5 and partitioning the methylation modifyingcompound between an aqueous phase and ethyl acetate above a pH of about10.
 156. The method according to claim 134, wherein the composition isessentially free of at least one substance selected from the groupconsisting of caffeine, caffeic acid and chlorogenic acid.
 157. Themethod according to claim 134, wherein in step (b) the composition isadministered orally.
 158. The method according to claim 134, wherein thecomposition is a powder.
 159. The method according to claim 134, whereinthe composition is in the form of a tablet.
 160. The method according toclaim 134, wherein the composition is in the form of a capsule.
 161. Themethod according to claim 134, wherein the composition is in the form ofa lozenge.
 162. The method according to claim 134, wherein thecomposition is in the form of a suppository.